Obesity related genes expressed at least in the hypothalamus, liver or pancreas

ABSTRACT

The present invention relates generally to nucleic acid molecules expressed at least in the hypothalamus, liver or pancreas identified using differential display techniques under differing physiological conditions. The nucleic acid molecules are associated with or act as markers for conditions of a healthy state, obesity, anorexia, weight maintenance, diabetes and/or metabolic energy levels. More particularly, the present invention is directed to a nucleic acid molecule and/or its expression product for use in therapeutic and diagnostic protocols for conditions such as obesity, anorexia, weight maintenance, diabetes and energy imbalance. The subject nucleic acid molecule and expression product and their derivatives, homologs, analogs and mimetics are proposed to be useful, therefore, as therapeutic and diagnostic agents for obesity, anorexia, weight maintenance, diabetes and energy imbalance or as targets for the design and/or identification of modulators of their activity and/or function.

FIELD OF THE INVENTION

The present invention relates generally to nucleic acid moleculesexpressed at least in the hypothalamus, liver or pancreas identifiedusing differential display techniques under differing physiologicalconditions. The nucleic acid molecules are associated with or act asmarkers for conditions of a healthy state, obesity, anorexia, weightmaintenance, diabetes and/or metabolic energy levels. More particularly,the present invention is directed to a nucleic acid molecule and/or itsexpression product for use in therapeutic and diagnostic protocols forconditions such as obesity, anorexia, weight maintenance, diabetes andenergy imbalance. The subject nucleic acid molecule and expressionproduct and their derivatives, homologs, analogs and mimetics areproposed to be useful, therefore, as therapeutic and diagnostic agentsfor obesity, anorexia, weight maintenance, diabetes and energy imbalanceor as targets for the design and/or identification of modulators oftheir activity and/or function.

BACKGROUND OF THE INVENTION

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

The increasing sophistication of recombinant DNA technology is greatlyfacilitating research and development in the medical, veterinary andallied human and animal health fields. This is particularly the case inthe investigation of the genetic bases involved in the etiology ofcertain disease conditions. One particularly significant condition fromthe stand point of morbidity and mortality is obesity and itsassociation with type 2 diabetes (formerly non-insulin-dependentdiabetes mellitus or NIDDM) and cardiovascular disease.

Obesity is defined as a pathological excess of body fat and is theresult of an imbalance between energy intake and energy expenditure fora sustained period of time. Obesity is the most common metabolic diseasefound in affluent nations. The prevalence of obesity in these nations isalarmingly high, ranging from 10% to upwards of 50% in somesubpopulations (Bouchard, The genetics of obesity. Boca Raton: CRCPress, 1994). Of particular concern is the fact that the prevalence ofobesity appears to be rising consistently in affluent societies and isnow increasing rapidly in less prosperous nations as they become moreaffluent and/or adopt cultural practices from the more affluentcountries (Zimmet, Diabetes Care 15(2): 232-247, 1992).

In 1995 in Australia, for example, 19% of the adult population wereobese (BMI>30). On average, women in 1995 weighed 4.8 kg more than theircounterparts in 1980 while men weighed 3.6 kg more (Australian Instituteof Health and Welfare (AIHW), Heart, Stroke and Vascular diseases,Australian facts. AIHW Cat. No. CVD 7 Canberra: AIHW and the HeartFoundation of Australia, 1999.). More recently, the AusDiab Studyconducted between the years 1999 and 2000 showed that 65% of males and45% of females aged 25-64 years were considered overweight (de Looperand Bhatia, Australia's Health Trends 2001. Australian Institute ofHealth and Welfare (AIHW) Cat. No. PHE 24. Canberra: AIHW, 2001). Theprevalence of obesity in the U.S. also increased substantially betweenand 1998, rising from 12% to 18% in Americans during this period (Mokdadet al., JAMA. 282(16): 1519-22, 1999).

The high and increasing prevalence of obesity has serious healthimplications for both individuals and society as a whole. Obesity is acomplex and heterogeneous disorder and has been identified as a key riskindicator of preventable morbidity and mortality since obesity increasesthe risk of a number of other metabolic conditions including type 2diabetes mellitus and cardiovascular disease (Must et al., JAMA.282(16): 1523-1529, 1999; Kopelman, Nature 404: 635-643, 2000).Alongside obesity, the prevalence of diabetes continues to increaserapidly. It has been estimated that there were about 700,000 personswith diabetes in Australia in 1995 while in the US, diabetes prevalenceincreased from 4.9% in 1990 to 6.9% in 1999 (Mokdad, Diabetes Care24(2): 412, 2001). In Australia, the annual costs of obesity associatedwith diabetes and other disease conditions has been conservativelyestimated to be AU$810 million for 1992-3 (National Health and MedicalResearch Council, Acting on Australia's weight: A strategy for theprevention of overweight and obesity. Canberra: National Health andMedical Research Council, 1996). In the US, the National HealthInterview Survey (NHIS) estimated the economic cost of obesity in 1995as approximately US$99 billion, thereby representing 5.7% of totalhealth costs in the U.S. at that time (Wolf and Colditz, Obes Res. 6:97-106, 1998).

A genetic basis for the etiology of obesity is indicated inter alia fromstudies in twins, adoption studies and population-based analyses whichsuggest that genetic effects account for 25-80% of the variation in bodyweight in the general population (Bouchard, 1994; supra; Kopelman etal., Int J Obesity 18: 188-191, 1994; Ravussin, Metabolisim 44(Suppl 3):12-14, 1995). It is considered that genes determine the possible rangeof body weight in an individual and then the environment influences thepoint within this range where the individual is located at any giventime (Bouchard, 1994; supra). However, despite numerous studies intogenes thought to be involved in the pathogenesis of obesity, there havebeen surprisingly few significant findings in this area. In addition,genome-wide scans in various population groups have not produceddefinitive evidence of the chromosomal regions having a major effect onobesity.

A number or organs/tissues have been implicated in the pathophysiologyof obesity and type 2 diabes. One organ of particular interest is thehypothalamus. Early studies led to the dual-center hypothesis whichproposed that two opposing centers in the hypothalamus were responsiblefor the initiation and termination of eating, the lateral hypothalamus(LHA; “hunger center”) and ventromedial hypothalamus (VMH; “satietycenter”; Stellar, Psycho. Rev. 61: 5-22, 1954). The dual-centerhypothesis has been repeatedly modified to accommodate the increasinginformation about the roles played by various other brain regions,neurotransmitter systems and hormonal and neural signals originating inthe gut on the regulation of food intake. In addition to the LHA andVMH, the paraventricular nucleus (PVN) is now considered to have animportant integrative function in the control of energy intake.

A large number of neurotransmitters has been investigated as possiblehypothalamic regulators of feeding behaviour including neuropeptide Y(NPY), glucagon-like peptide 1 (GLP-1), melanin-concentrating hormone(MCH), serotonin, cholecystokinin and galanin. Some of theseneurotransmitters stimulate food intake, some act in an anorexigenicmanner and some have diverse effects on energy intake depending on thesite of administration.

For example, γ-aminobutyric acid (GABA) inhibits food intake wheninjected into the LHA, but stimulates eating when injected into the VMHor PVN (Leibowitz, Fed. Proc. 45(5): 1396-403, 1985). Feeding behaviouris thought to be greatly influenced by the interaction of stimulatoryand inhibitory signals in the hypothalamus.

Another organ of interest is the liver.

The liver plays a significant role in a number of importantphysiological pathways. It has a major role in the regulation ofmetabolism of glucose, amino acids and fat. In addition the liver is theonly organ (other than the gut) that comes into direct contact with alarge volume of ingested food and therefore the liver is able to “sense”or monitor the level of nutrients entering the body, particularly theamounts of protein and carbohydrate. It has been proposed that the livermay also have a role in the regulation of food intake through thetransmission of unidentified signals relaying information to the brainabout nutrient absorption from the gut and metabolic changes throughoutthe body (Russek, Nature 200 176, 1963; Koopmans, “Experimental studieson the control of food intake”. In: Handbook of Obesity, Eds. G. A.Bray, C. Bouchard, W. P. T. Gray, pp. 273-312, 1998). The liver alsoplays a crucial role in maintaining circulating glucose concentrationsby regulating pathways such as gluconeogenesis and glycogenolysis.Alterations in glucose homeostasis are important factors in thepathophysiology of impaired glucose tolerance and the development oftype 2 diabetes mellitus.

In accordance with the present invention, genetic sequences were soughtwhich are differentially expressed in lean and obese animals or in fedcompared to unfed animals. Novel genes are identified which are proposedto be associated with or act as markers for energy balance as well as ahealthy state, obesity, anorexia, weight maintenance and diabetes.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A sequence listing is provided after the claims.

Differential display analysis of genetic material from hypothalamus,liver and pancreatic tissue were used to identify candidate geneticsequences associated with a healthy state or with physiologicalconditions such as obesity, anorexia, weight maintenance, diabetesand/or metabolic energy levels. An animal model was employed comprisingthe Israeli Sand Rat (Psammomys obesus). Three groups of animals wereused designated Groups A, B and C based on metabolic phenotype asfollows:—

-   Group A: lean animals (normoglycemic; normoinsulinemic);-   Group B: obese, non-diabetic animals (normoglycemic;    hyperinsulinemic); and-   Group C: obese, diabetic animals (hyperglycemic; hyperinsulinemic).

Animals were maintained under fed or unfed conditions or underconditions of high or low glucose or insulin and genetic sequencesanalyzed by differential display analysis. In a preferred embodimentusing these techniques, six differentially expressed sequences wereidentified from hypothalamus cells designated herein AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 with sequence identifiers SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ IDNO:6, respectively.

AGT-109 was detected initially in hypothalamus tissue using differentialdisplay PCR and its expression was elevated in fasted Group A and Banimals compared to fed animals. AGT-407 was initially detected in liverusing suppression subtractive hybridization (SSH) and its expression waselevated in Group A animals in a fasted state compared to Group B and Canimals under similar conditions. Consequently, this gene is expressedin healthy animals compared to obese or diabetic animals. AGT-408 wasinitially identified in the liver using SSH and its expression levelswere lower in fed, healthy animals, i.e. Group A animals, compared tofasted Group A animals or fed Group B animals. AGT-409 was initiallyidentified in the liver using SSH and was shown to have elevatedexpression levels in fed, healthy animals, i.e. Group A animals,compared to Group A, B or C animals under fasting conditions. AGT-601was identified in silico in hypothalamus tissue and its expression waselevated in diabetic, obese animals, i.e. Group C animals, compared toother groups. In general, the expression of this gene was elevated infed animals compared to fasting animals regardless of which group.AGT-204 was identified in the pancreas using differential expressionanalysis and its expression was found to be elevated in fed compared tofasting animals. A summary of the AGT genes is provided in Table 1.

Table 1 Summary of Differentially Expressed Genes

TABLE 1 Summary of Differentially Expressed Genes METHOD SEQ ID OF GENENO: TISSUE PHENOTYPE DETECTION AGT-109 1 Hypothalamus Expressionelevated in healthy Differential (Group A) and diabetic, non- displayPCR obese (Group B) animals compared to fed animals AGT-407 2 LiverExpression levels elevated in Suppression fasted Group A animalssubtractive compared to Group B and hybridization diabetic, obese (GroupC) (SSH) animals AGT-408 3 Liver Expression levels lower in fed SSHGroup A animals compared to fasted Group A animals or fed Group Banimals AGT-409 4 Liver Expression levels elevated in fed SSH Group Aanimals compared to fasting Groups A, B or C animals AGT-601 5Hypothalamus Expression levels elevated in fed in silico Group C animalscompared to differential fed Groups A or B animals. In expressiongeneral, elevated expression in fed versus fasting animals. AGT-204 6Pancreas Expression levels elevated in fed Differential compared tofasting animals display

The identification of these variably expressed sequences permits therationale design and/or selection of molecules capable of antagonizingor agonizing the expression products and/or permits the development ofscreening assays. The screening assays, for example, include assessingthe physiological status of a particular subject.

Accordingly, one aspect of the present invention provides a nucleic acidmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding a protein or mRNA or a derivative, homolog,analog or mimetic thereof wherein the nucleic acid molecule isdifferentially expressed in hypothalamus, liver or pancreas betweenfasted and fed animals and/or between diabetic and non-diabetic animals.

In a preferred embodiment, the nucleic acid molecule comprises anucleotide sequence substantially as set forth in SEQ ID NO: 1 or SEQ IDNO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or anucleotide sequence having at least about 30% similarity to all or partof SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ IDO NO:4 or SEQ IDNO:5 or SEQ ID NO:6 and/or is capable of hybridizing to one or more ofSEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5or SEQ ID NO:6 or their complementary forms under low stringencyconditions.

Another aspect of the present invention provides an isolated molecule ora derivative, homolog, analog or mimetic thereof which is produced indifferential amounts in hypothalamus, liver or pancreas tissue of obeseanimals compared to lean animals and/or in hypothalamus, liver orpancreas tissue of fasted animals compared to fed animals.

The molecule is generally a protein but may also be an mRNA, intron orexon. In this respect, the molecule may be considered an expressionproduct of the subject nucleotide sequences.

In a preferred embodiment, the nucleic acid molecule comprises anucleotide sequence substantially as set forth in SEQ ID NO:1 or SEQ IDNO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6.

The preferred genetic sequence of the present invention are referred toherein as AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204. Theexpression products encoded by AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and AGT-204 are referred to herein as AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204, respectively. The expression product maybe an RNA (e.g. mRNA) or a protein. Where the expression product is anRNA, the present invention extends to RNA-related molecules associatedthereto such as RNAi.

A further aspect of the present invention relates to a compositioncomprising AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 orits derivatives, homologs, analogs or mimetics or agonists orantagonists of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204together with one or more pharmaceutically acceptable carriers and/ordiluents.

Yet a further aspect of the present invention contemplates a method fortreating a subject comprising administering to said subject a treatmenteffective amount of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 or a derivative, homolog, analog or mimetic thereof or a geneticsequence encoding same or an agonist or antagonist of AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 activity or AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 gene expression for a time andunder conditions sufficient to effect treatment.

In accordance with this and other aspects of the present invention,treatments contemplated herein include but are not limited to obesity,anorexia, weight maintenance, energy imbalance and diabetes. Treatmentmay be by the administration of a pharmaceutical composition or geneticsequences via gene therapy. Treatment is contemplated for human subjectsas well as animals such as animals important to livestock industry.

Still yet another aspect of the present invention is directed to adiagnostic agent for use in monitoring or diagnosing conditions such asbut not limited to obesity, anorexia, weight maintenance, energyimbalance and/or diabetes, said diagnostic agent selected from anantibody to AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 orits derivatives, homologs, analogs or mimetics and a genetic sequencecomprising or capable of annealing to a nucleotide strand associatedwith AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 usefulinter alia in PCR, hybridization and/or RFLP.

A summary of sequence identifiers used throughout the subjectspecification is provided in Table 2. TABLE 2 Summary of SequenceIdentifiers SEQUENCE ID NO. DESCRIPTION 1 partial nucleotide sequence ofAGT-109 2 partial nucleotide sequence of AGT-407 3 partial nucleotidesequence of AGT-408 4 partial nucleotide sequence of AGT-409 5 partialnucleotide sequence of AGT-601 6 partial nucleotide sequence of AGT-2047 AGT-109 forward primer 8 AGT-109 reverse primer 9 AGT-407 forwardprimer 10 AGT-407 reverse primer 11 AGT-408 forward primer 12 AGT-408reverse primer 13 AGT-409 forward primer 14 AGT-409 reverse primer 15AGT-204 forward primer 16 AGT-204 reverse primer 17 AGT-601 forwardprimer 18 AGT-601 reverse primer 19 AGT-601 probe 20 β-actin forwardprimer 21 β-actin reverse primer 22 β-actin probe 23 Cyclophilin forwardprimer 24 Cyclophilin reverse primer 25 Cyclophilin probe

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of AGT-109 express in thehypothalamus of fed and fasted animals.

FIG. 2 is a graphical representation of AGT-109 expression in thehypothalamus of fed and fasted animals (pooled animal data). *p<0.001.

FIG. 3 is a graphical representation of hypothalamic AGT-109 expression.

FIG. 4 is a graphical representation of AGT-407 expression in the liverof fed and fasted animals.

FIG. 5 is a graphical representation of AGT-407 expression in the liverof fed and fasted animals (pooled animal data). *p<0.003.

FIG. 6 is a schematic representation of the genomic structure of the S1Pgene.

FIG. 7 is a schematic representation of the relationship between exonorganization and functional domains of S1P (Nakajima et al., J. Hum.Genet. 45: 212-217, 2000).

FIG. 8 is a graphical representation of AGT-408 expression in the liversof fed and fasted animals.

FIG. 9 is a graphical representation of AGT-408 expression in the liverof fed and fasted animals (pooled animal data).

FIG. 10 is a graphical representation of AGT-409 expression in the liverof fed and fasted animals.

FIG. 11 is a graphical representation of AGT-409 expression in the liverof fed and fasted animals (pooled animal data). *p<0.001.

FIG. 12 is a graphical representation of AGT-601 expression in thehypothalamus of fed and fasted animals. * Significantly different from Afed and B fed groups, p=0.004 and p=0.005, respectively. {circumflexover ( )} Significantly different from C fasted group, p=0.001.

FIG. 13 is a graphical representation of AGT-601 in the hypothalamus offed and fasted animals (pooled animal data). *p=0.015

FIG. 14 is a graphical representation of the Log AGT-601 versus Logglucose of fed animals.

FIG. 15 is a graphical representation of the Log AGT-601 versus % bodyfat of fed animals.

FIG. 16 is a graphical representation of AGT-601 gene expression in thepresence of saline and beacon (see PCT/AU98/00902 [WO 99/23217]). *p=0.03, significantly different to saline group. ** p=0.004,significantly different to NPY+Beacon group. # p=0.005, significantlydifferent to NPY+Beacon group.

FIG. 17 is a graphical representation of AGT-601 expression ininsulin-treated GT17 cells.

FIG. 18 is a graphical representation of AGT-601 expression inglucose-treated GT17 cells.

FIG. 19 is a graphical representation of AGT-204 expression in thepancreas of fed and fasted animals.

FIG. 20 is a graphical representation of AGT-204 expression in thepancreas of fed and asted animals (pooled animal data). *p=0.001.

FIG. 21 is a graphical representation of hypothalamus AGT-204 expressionunder fed or fasting conditions. * p=0.05 Group A fed versus Group Afasted animals; # p=0.03 Group B fed versus B fasted animals.

FIG. 22 is a graphical representation of hypothalamus AGT-204 expressionof all animals under fed and fasting conditions. * p=0.009.

FIG. 23 is a graphical representation of hypothalamus AGT-204 expressionin control and restricted animals. * p<0.03, significantly different toGroup A control.

FIG. 24 is a graphical representation of control body weight (BW) andAGT-204 expression.

FIG. 25 is a

FIG. 26 is a schematic representation of the hybridization andamplification stages of the SSH (RDA) protocol.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the identification ofnovel genes associated inter alia with regulation of energy balanceobesity and diabetes and/or muscle development. The genes wereidentified by a number of procedures including differential display,microarray analysis or suppression subtractive hybridization (SSH) [alsoreferred to as representative difference analysis (RDA)] ofhypothalamus, liver or pancreas mRNA between lean and obese animalsand/or between fed animals and fasted animals and/or between diabeticand non-diabetic animals.

The term “differential” array is used in its broadest sense to includethe expression of nucleic acid sequences in one type of tissue relativeto another type of tissue in the same or different animals. Reference to“different” animals includes the same animals but in differentgastronomical states such as in a fed or non-fed state. A microarrayanalysis preferably includes sets of arrays of nucleic acid expressionproducts (e.g. mRNA or PCR products) which display differentialhybridization characteristics.

Accordingly, one aspect of the present invention provides a nucleic acidmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding a protein or a derivative, homolog, analog ormimetic thereof wherein said nucleic acid molecule is expressed inlarger or smaller amounts in hypothalamus, liver and/or pancreas ofobese animals compared to lean animals.

In a related embodiment, the present invention provides a nucleic acidmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding a protein or a derivative, homolog, analog ormimetic thereof wherein said nucleic acid molecule is expressed inlarger or smaller amounts in the hypothalamus, liver and/or pancreas offed animals compared to fasted animals.

In yet another related embodiment, the present invention provides anucleic acid molecule comprising a sequence of nucleotides encoding orcomplementary to a sequence encoding a protein or a derivative, homolog,analog or mimetic thereof wherein said nucleic acid molecule isexpressed in larger or smaller amounts in the hypothalamus, liver and/orpancreas of diabetic animals compared to non-diabetic animals.

The terms “lean” and “obese” are used in their most general sense butshould be considered relative to the standard criteria for determiningobesity. Generally, for human subjects, the definition of obesity isBMI>30 (Risk Factor Prevalence Study Management Committee. Risk FactorPrevalance Study: Survey No. 3:1989. Canberra: National hearthFoundation of Australia and Australian Institute of Health, 1990; Watersand Bennett, Risk Factors for Cardiovascular Disease: A Summary ofAustralian data. Canberra: Australian Institute of Health and Welfare,1995).

Conveniently, an animal model may be employed to study the differencesin gene expression between obese and lean animals and fasted and fedanimals. In particular, the present invention is exemplified using thePsammomys obesus (the Israeli sand rat) animal model of dietary-inducedobesity and NIDDM. In its natural desert habitat, an active lifestyleand saltbush diet ensure that they remain lean and normoglycemic(Shafrir and Gutman, J Basic Clin Physiol Pharm 4: 83-99, 1993).However, in a laboratory setting on a diet of ad libitum chow (on whichmany other animal species remain healthy), a range of pathophysiologicalresponses are seen (Barnett et al., Diabetologia 37: 671-676, 1994a;Barnett et al., Int. J. Obesity 18: 789-794, 1994b, Barnett et al.,Diabete Nutr Metab 8: 42-47, 1995). By the age of 16 weeks, more thanhalf of the animals become obese and approximately one-third developNIDDM. Only hyperphagic animals go on to develop hyperglycemia,highlighting the importance of excessive energy intake in thepathophysiology of obesity and NIDDM in Psammomys obesus (Collier etal., Ann New York Acad Sci 827: 50-63, 1997a; Walder et al., Obesity Res5: 193-200, 1997a). Other phenotypes found include hyperinsulinemia,dyslipidemia and impaired glucose tolerance (Collier et al., 1997a;supra; Collier et al., Exp Clin Endocrinol Diabetes 105: 36-37, 1997b).Psammomys obesus exhibit a range of bodyweight and blood glucose andinsulin levels which forms a continuous curve that closely resembles thepatterns found in human populations, including the inverted U-shapedrelationship between blood glucose and insulin levels known as“Starling's curve of the pancreas” (Barnett et al., 1994a; supra). It isthe heterogeneity of the phenotypic response of Psammomys obesus whichmake it an ideal model to study the etiology and pathophysiology ofobesity and NIDDM.

Psammomys obesus animals are conveniently divided into three groups vizGroup A animals which are lean, normoglycemic and normoinsulinemic,Group B animals which are obese, normoglycemic and hyperinuslinemic andGroup C animals which are obese, hyperglycemic and hyperinsulinemic.

Another aspect of the present invention provides a nucleic acid moleculecomprising a nucleotide sequence encoding or complementary to a sequenceencoding an expression product wherein said nucleotide sequence is assubstantially set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 orSEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or a nucleotide sequencehaving at least about 30% similarity to all or part of SEQ ID NO:1 orSEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6and/or is capable of hybridizing to one or more of SEQ ID NO:1 or SEQ IDNO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 ortheir complementary forms under low stringency conditions at 42° C. andwherein said nucleic acid molecule is expressed in larger or smalleramounts in hypothalamus, liver or pancreas of obese animals compared tolean animals and/or in fed animals compared to fasted animals.

Higher similarities are also contemplated by the present invention suchas greater than 40% or 50% or 60% or 70% or 80% or 90% or 95% or 96% or97% or 98% or 99% or above.

An expression product includes an RNA molecule such as a mRNA transcriptas well as a protein. Some genes are non-protein encoding genes andproduce mRNA or other RNA type molecules and are involved in regulationby RNA:DNA, RNA:RNA or RNA:protein interaction. The RNA (e.g. mRNA) mayact directly or via the induction of other molecules such as RNAi or viaproducts mediated from splicing events (e.g. exons or introns). Othergenes encode mRNA transcripts which are then translated into proteins. Aprotein includes a polypeptide. The differentially expressed nucleicacid molecules, therefore, may encode mRNAs only or, in addition,proteins. Both mRNAs and proteins are forms of “expression products”.

Reference herein to similarity is generally at a level of comparison ofat least 15 consecutive or substantially consecutive nucleotides or atleast 5 consecutive or substantially consecutive amino acid residues.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide or amino acid level. Where there isnon-identity at the nucleotide level, “similarity” includes differencesbetween sequences which result in different amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. In a particularly preferredembodiment, nucleotide and sequence comparisons are made at the level ofidentity rather than similarity.

Terms used to describe sequence relationships between two or morepolynucleotides include “reference sequence”, “comparison window”,“sequence similarity”, “sequence identity”, “percentage of sequencesimilarity”, “percentage of sequence identity”, “substantially similar”and “substantial identity”. A “reference sequence” is at least 12 butfrequently 15 to 18 and often at least 25 or above, such as 30 monomerunits in length. Because two polynucleotides may each comprise (1) asequence (i.e. only a portion of the complete polynucleotide sequence)that is similar between the two polynucleotides, and (2) a sequence thatis divergent between the two polynucleotides, sequence comparisonsbetween two (or more) polynucleotides are typically performed bycomparing sequences of the two polynucleotides over a “comparisonwindow” to identify and compare local regions of sequence similarity. A“comparison window” refers to a conceptual segment of typically 12contiguous residues that is compared to a reference sequence. Thecomparison window may comprise additions or deletions (i.e. gaps) ofabout 20% or less as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by computerised implementations of algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Drive Madison,Wis., USA) or by inspection and the best alignment (i.e. resulting inthe highest percentage homology over the comparison window) generated byany of the various methods selected. Reference also may be made to theBLAST family of programs as for example disclosed by Altschul et al.(Nucl. Acids Res. 25: 3389, 1997). A detailed discussion of sequenceanalysis can be found in Unit 19.3 of Ausubel et al. (“Current Protocolsin Molecular Biology” John Wiley & Sons Inc, 1994-1998, Chapter 15).

The terms “sequence similarity” and “sequence identity” as used hereinrefers to the extent that sequences are identical or functionally orstructurally similar on a nucleotide-by-nucleotide basis over a windowof comparison. Thus, a “percentage of sequence identity”, for example,is calculated by comparing two optimally aligned sequences over thewindow of comparison, determining the number of positions at which theidentical nucleic acid base (e.g. A, T, C, G, I) occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. For the purposes of thepresent invention, “sequence identity” will be understood to mean the“match percentage” calculated by the DNASIS computer program (Version2.5 for windows; available from Hitachi Software engineering Co., Ltd.,South San Francisco, Calif., USA) using standard defaults as used in thereference manual accompanying the software. Similar comments apply inrelation to sequence similarity.

Reference herein to a low stringency includes and encompasses from atleast about 0 to at least about 15% v/v formamide and from at leastabout 1 M to at least about 2 M salt for hybridization, and at leastabout 1 M to at least about 2 M salt for washing conditions. Generally,low stringency is at from about 25-30° C. to about 42° C. Thetemperature may be altered and higher temperatures used to replaceformamide and/or to give alternative stringency conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization, and at least about 0.5 M to atleast about 0.9 M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% v/v to at least about50% v/v formamide and from at least about 0.01 M to at least about 0.15M salt for hybridization, and at least about 0.01 M to at least about0.15 M salt for washing conditions. In general, washing is carried outT_(m)=69.3±0.41 (G+C)% (Marmur and Doty, J. Mol. Biol. 5: 109, 1962).However, the T_(m) of a duplex DNA decreases by 1° C. with everyincrease of 1% in the number of mismatch base pairs (Bonner and Laskey,Eur. J. Biochem. 46: 83, 1974. Formamide is optional in thesehybridization conditions. Accordingly, particularly preferred levels ofstringency are defined as follows: low stringency is 6×SSC buffer, 0.1%w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/vSDS at a temperature in the range 20° C. to 65° C.; high stringency is0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.

The nucleotide sequence or amino acid sequence of the present inventionmay correspond to exactly the same sequence of the naturally occurringgene (or corresponding cDNA) or protein or may carry one or morenucleotide or amino acid substitutions, additions and/or deletions. Thenucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 correspond to the genesreferred to herein as AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204, respectively. The corresponding proteins are AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204, respectively. Reference herein toAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 includes, whereappropriate, reference to the genomic gene or cDNA as well as anynaturally occurring or induced derivatives. Apart from thesubstitutions, deletions and/or additions to the nucleotide sequence,the present invention further encompasses mutants, fragments, parts andportions of the nucleotide sequence corresponding to AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204.

Another aspect of the present invention provides a nucleic acid moleculeor derivative, homolog or analog thereof comprising a nucleotidesequence encoding, or a nucleotide sequence complementary to anucleotide sequence encoding, an amino acid sequence substantially asset forth in SEQ ID NO:1 or a derivative, homolog or mimetic thereof orhaving at least about 30% similarity to at least 10 contiguous aminoacids in SEQ ID NO:1.

Yet another aspect of the present invention provides a nucleic acidmolecule or derivative, homolog or analog thereof comprising anucleotide sequence encoding, or a nucleotide sequence complementary toa nucleotide sequence encoding, an amino acid sequence substantially asset forth in SEQ ID NO:2 or a derivative, homolog or mimetic thereof orhaving at least about 30% similarity to at least 10 contiguous aminoacids in SEQ ID NO:2.

Still yet another aspect of the present invention provides a nucleicacid molecule or derivative, homolog or analog thereof comprising anucleotide sequence encoding, or a nucleotide sequence complementary toa nucleotide sequence encoding, an amino acid sequence substantially asset forth in SEQ ID NO:3 or a derivative, homolog or mimetic thereof orhaving at least about 30% similarity to at least 10 contiguous aminoacids in SEQ ID NO:3.

Even still another aspect of the present invention provides a nucleicacid molecule or derivative, homolog or analog thereof comprising anucleotide sequence encoding, or a nucleotide sequence complementary toa nucleotide sequence encoding, an amino acid sequence substantially asset forth in SEQ ID NO:4 or a derivative, homolog or mimetic thereof orhaving at least about 30% similarity to at least 10 contiguous aminoacids in SEQ ID NO:4.

Even yet another aspect of the present invention provides a nucleic acidmolecule or derivative, homolog or analog thereof comprising anucleotide sequence encoding, or a nucleotide sequence complementary toa nucleotide sequence encoding, an amino acid sequence substantially asset forth in SEQ ID NO:5 or a derivative, homolog or mimetic hereof orhaving at least about 30% similarity to at least 10 contiguous aminoacids in SEQ ID NO:5.

Even yet another aspect of the present invention provides a nucleic acidmolecule or derivative, homolog or analog thereof comprising anucleotide sequence encoding, or a nucleotide sequence complementary toa nucleotide sequence encoding, an amino acid sequence substantially asset forth in SEQ ID NO:6 or a derivative, homolog or mimetic thereof orhaving at least about 30% similarity to at least 10 contiguous aminoacids in SEQ ID NO:6.

The expression pattern of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601and AGT-204 has been determined, inter alia, to indicate an involvementin the regulation of one or more of obesity, diabetes and/or energymetabolism. In addition to the differential expression of AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 in the muscle,hypothalamus, liver, stomach and/or pancreas of lean versus obeseanimals and fed versus fasted animals, these genes may also be expressedin other tissues including but in no way limited to muscle,hypothalamus, liver, stomach and/or pancreas. The nucleic acid moleculeencoding each of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204is preferably a sequence of deoxyribonucleic acids such as a cDNAsequence or a genomic sequence. A genomic sequence may also compriseexons and introns. A genomic sequence may also include a promoter regionor other regulatory regions.

A homolog is considered to be a AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and AGT-204 gene from another animal species. The AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 genes are exemplifiedherein from the hypothalamus, liver and/or the pancreas of Psammomysobesus. The invention extends, however, to the homologous genes, asdetermined by nucleotide sequence and/or function, from humans,primates, livestock animals (e.g. cows, sheep, pigs, horses, donkeys),laboratory test animals (e.g. mice, guinea pigs, hamsters, rabbits),companion animals (e.g. cats, dogs) and captured wild animals (e.g.rodents, foxes, deer, kangaroos).

The nucleic acids of the present invention and in particular AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 and their derivatives andhomologs may be in isolated or purified from and/or may be ligated to avector such as an expression vector. Expression may be in a eukaryoticcell line (e.g. mammalian, insect or yeast cells) or in microbial cells(e.g. E. coli) or both.

The derivatives of the nucleic acid molecules of the present inventioninclude oligonucleotides, PCR primers, antisense molecules, moleculessuitable for use in co-suppression and fusion nucleic acid molecules.Ribozymes and DNA enzymes are also contemplated by the present inventiondirected to AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 ortheir mRNAs. Derivatives and homologs of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 are conveniently encompassed by thosenucleotide sequences capable of hybridizing to SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 under lowstringency conditions at 42° C.

Derivatives include fragments, parts, portions, mutants, variants andmimetics from natural, synthetic or recombinant sources including fusionproteins. Parts or fragments include, for example, active regions ofAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204. Derivatives maybe derived from insertion, deletion or substitution of amino acids.Amino acid insertional derivatives include amino and/or carboxylicterminal fusions as well as intrasequence insertions of single ormultiple amino acids. Insertional amino acid sequence variants are thosein which one or more amino acid residues are introduced into apredetermined site in the protein although random insertion is alsopossible with suitable screening of the resulting product. Deletionalvariants are characterized by the removal of one or more amino acidsfrom the sequence. Substitutional amino acid variants are those in whichat least one residue in the sequence has been removed and a differentresidue inserted in its place. An example of substitutional amino acidvariants are conservative amino acid substitutions. Conservative aminoacid substitutions typically include substitutions within the followinggroups: glycine and alanine; valine, isoleucine and leucine; asparticacid and glutamic acid; asparagine and glutamine; serine and threonine;lysine and arginine; and phenylalanine and tyrosine. Additions to aminoacid sequences include fusions with other peptides, polypeptides orproteins.

Chemical and functional equivalents of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 should be understood as moleculesexhibiting any one or more of the functional activities of thesemolecules and may be derived from any source such as being chemicallysynthesized or identified via screening processes such as naturalproduct screening.

The derivatives include fragments having particular epitopes or parts ofthe entire protein fused to peptides, polypeptides or otherproteinaceous or non-proteinaceous molecules.

Another aspect of the present invention provides an isolated protein ora derivative, homolog, analog or mimetic thereof which is produced inlarger or smaller amounts in the hypothalamus, liver and/or pancreas ofin obese animals compared to lean animals.

In a more preferred aspect of the present invention, there is providedan isolated protein or a derivative, homolog, analog or mimetic thereofwherein said protein comprises an amino acid sequence substantiallyencoded by a nucleotide sequence as set forth in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 or an aminoacid sequence having at least 30% similarity to all or part thereof andwherein said protein is produced in larger or smaller amounts in liveror stomach of obese animals compared to lean animals.

A further aspect of the present invention is directed to an isolatedprotein or a derivative, homolog, analog or mimetic thereof wherein saidprotein is encoded by a nucleotide sequence substantially as set forthin SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 orSEQ ID NO:6 or a nucleotide sequence having at least 60% similarity toall or part of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5 or SEQ ID NO:6 and/or is capable of hybridizing to SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 ortheir complementary forms under low stringency conditions at 42° C.

Reference herein to AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 includes reference to isolated or purified naturally occurringAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 proteinmolecules as well as any derivatives, homologs, analogs and mimeticsthereof. Derivatives include parts, fragments and portions of AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 as well as single andmultiple amino acid substitutions, deletions and/or additions toAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204. A derivative ofAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 is convenientlyencompassed by molecules encoded by a nucleotide sequence capable ofhybridizing to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4or SEQ ID NO:5 or SEQ ID NO:6 under low stringency conditions at 42° C.

Other derivatives of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 include chemical analogs. Analogs of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 contemplated herein include, but are notlimited to, modifications to side chains, incorporation of unnaturalamino acids and/or their derivatives during peptide, polypeptide orprotein synthesis and the use of crosslinkers and other methods whichimpose conformational constraints on the proteinaceous molecule or theiranalogs.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzenesulphonic acid (TNBS); acylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitization, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids. A list of unnaturalamino acid, contemplated herein is shown in Table 3. TABLE 3 Codes fornon-convention amino acids Non-conventional amino acid CodeNon-conventional amino acid Code α-aminobutyric acid AbuL-N-methylalanine Nmala α-amino-α-methylbutyrate MgabuL-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagineNmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilize 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, peptides can beconformationally constrained by, for example, incorporation of Cα and Nα-methylamino acids, introduction of double bonds between Cα and Cβatoms of amino acids and the formation of cyclic peptides or analogs byintroducing covalent bonds such as forming an amide bond between the Nand C termini, between two side chains or between a side chain and the Nor C terminus.

All such modifications may also be useful in stabilizing the AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 molecule for use in invivo administration protocols or for diagnostic purposes.

The nucleic acid molecule of the present invention is preferably inisolated form or ligated to a vector, such as an expression vector. By“isolated” is meant a nucleic acid molecule having undergone at leastone purification step and this is conveniently defined, for example, bya composition comprising at least about 10% subject nucleic acidmolecule, preferably at least about 20%, more preferably at least about30%, still more preferably at least about 40-50%, even still morepreferably at least about 60-70%, yet even still more preferably 80-90%or greater of subject nucleic acid molecule relative to other componentsas determined by molecular weight, encoding activity, nucleotidesequence, base composition or other convenient means. The nucleic acidmolecule of the present invention may also be considered, in a preferredembodiment, to be biologically pure.

The term “protein” should be understood to encompass peptides,polypeptides and proteins. The protein may be glycosylated orunglycosylated and/or may contain a range of other molecules fused,linked, bound or otherwise associated to the protein such as aminoacids, lipids, carbohydrates or other peptides, polypeptides orproteins. Reference hereinafter to a “protein” includes a proteincomprising a sequence of amino acids as well as a protein associatedwith other molecules such as amino acids, lipids, carbohydrates or otherpeptides, polypeptides or proteins.

In a particularly preferred embodiment, the nucleotide sequencecorresponding to AGT-109 is a cDNA sequence comprising a sequence ofnucleotides as set forth in SEQ ID NO:1 or a derivative, homolog oranalog thereof including a nucleotide sequence having similarity to SEQID NO:1.

In another particularly preferred embodiment, the nucleotide sequencecorresponding to AGT-407 is a cDNA sequence comprising a sequence ofnucleotides as set forth in SEQ ED NO:2 or a derivative, homolog oranalog thereof including a nucleotide sequence having similarity to SEQID NO:2.

In still another particularly preferred embodiment, the nucleotidesequence corresponding to AGT-408 is a cDNA sequence comprising asequence of nucleotides as set forth in SEQ ID NO:3 or a derivative,homolog or analog thereof including a nucleotide sequence havingsimilarity to SEQ ID NO:3.

In a further particularly preferred embodiment, the nucleotide sequencecorresponding to AGT-409 is a cDNA sequence comprising a sequence ofnucleotides as set forth in SEQ ID NO:4 or a derivative, homolog oranalog thereof including a nucleotide sequence having similarity to SEQID NO:4.

In still a further particularly preferred embodiment, the nucleotidesequence corresponding to AGT-01 is a cDNA sequence comprising asequence of nucleotides as set forth in SEQ ID NO:5 or a derivative,homolog or analog thereof including a nucleotide sequence havingsimilarity to SEQ ID NO:5.

In still a further particularly preferred embodiment, the nucleotidesequence corresponding to AGT-204 is a cDNA sequence comprising asequence of nucleotides as set forth in SEQ ID NO:6 or a derivative,homolog or analog thereof including a nucleotide sequence havingsimilarity to SEQ ID NO:6.

The nucleic acid molecule may be ligated to an expression vector capableof expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell(e.g. yeast cells, fungal cells, insect cells, mammalian cells or plantcells). The nucleic acid molecule may be ligated or fused or otherwiseassociated with a nucleic acid molecule encoding another entity such as,for example, a signal peptide. It may also comprise additionalnucleotide sequence information fused, linked or otherwise associatedwith it either at the 3′ or 5′ terminal portions or at both the 3′ and5′ terminal portions. The nucleic acid molecule may also be part of avector, such as an expression vector.

The present invention extends to the expression product of the nucleicacid molecules as hereinbefore defined.

Preferably, the expression products are AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 having an amino acid sequence encoded bySEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQID NO:6, respectively or are derivatives, analogs, homologs, chemicalequivalents or mimetics thereof.

Another aspect of the present invention is directed to an isolatedprotein selected from the list consisting of:—

-   (i) a protein encoded by a novel nucleic acid molecule which    molecule is differentially expressed in hypothalamus, liver and/or    pancreas of obese animals compared to lean animals or a derivative,    homolog, analog, chemical equivalent or mimetic thereof;-   (ii) a protein encoded by a novel nucleic acid molecule which    molecule is differentially expressed in hypothalamus, liver and/or    pancreas of fed animals compared to fasted animals or a derivative,    homolog, analog, chemical equivalent or mimetic thereof;-   (iii) a protein encoded by a nucleotide sequence substantially as    set forth in SEQ ID NO:1 or a derivative, homolog or analog thereof    or a sequence encoding an amino acid sequence having at least about    45% similarity to this sequence or a derivative, homolog, analog,    chemical equivalent or mimetic of said protein;-   (iv) a protein encoded by a nucleotide sequence substantially as set    forth in SEQ ID NO:2 or a derivative, homolog or analog thereof or a    sequence encoding an amino acid sequence having at least about 45%    similarity to this sequence or a derivative, homolog, analog,    chemical equivalent or mimetic of said protein;-   (v) a protein encoded by a nucleotide sequence substantially as set    forth in SEQ ID NO:3 or a derivative, homolog or analog thereof or a    sequence encoding an amino acid sequence having at least about 45%    similarity to this sequence or a derivative, homolog, analog,    chemical equivalent or mimetic of said protein;-   (vi) a protein encoded by a nucleotide sequence substantially as set    forth in SEQ ID NO:4 or a derivative, homolog or analog thereof or a    sequence encoding an amino acid sequence having at least about 45%    similarity to this sequence or a derivative, homolog, analog,    chemical equivalent or mimetic of said protein;-   (vii) a protein encoded by a nucleotide sequence substantially as    set forth in SEQ ID NO:5 or a derivative, homolog or analog thereof    or a sequence encoding an amino acid sequence having at least about    45% similarity to this sequence or a derivative, homolog, analog,    chemical equivalent or mimetic of said protein;-   (viii) a protein encoded by a nucleotide sequence substantially as    set forth in SEQ ID NO:6 or a derivative, homolog or analog thereof    or a sequence encoding an amino acid sequence having at least about    45% similarity to this sequence or a derivative, homolog, analog,    chemical equivalent or mimetic of said protein;-   (ix) a protein encoded by a nucleic acid molecule capable of    hybridizing to the nucleotide sequence as set forth in SEQ ID NO:1    or a derivative, homolog or analog thereof under low stringency    conditions;-   (x) a protein encoded by a nucleic acid molecule capable of    hybridizing to the nucleotide sequence as set forth in SEQ ID NO:2    or a derivative, homolog or analog thereof under low stringency    conditions;-   (xi) a protein encoded by a nucleic acid molecule capable of    hybridizing to the nucleotide sequence as set forth in SEQ ID NO:3    or a derivative, homolog or analog thereof under low stringency    conditions;-   (xii) a protein encoded by a nucleic acid molecule capable of    hybridizing to the nucleotide sequence as set forth in SEQ ID NO:4    or a derivative, homolog or analog thereof under low stringency    conditions;-   (xiii) a protein encoded by a nucleic acid molecule capable of    hybridizing to the nucleotide sequence as set forth in SEQ ID NO:5    or a derivative, homolog or analog thereof under low stringency    conditions;-   (xiv) a protein encoded by a nucleic acid molecule capable of    hybridizing to the nucleotide sequence as set forth in SEQ ID NO:6    or a derivative, homolog or analog thereof under low stringency    conditions;-   (xv) a protein as defined in any one of paragraphs (i) to (xiv) in a    homodimeric form;-   (xvi) a protein as defined in any one of paragraphs (i) to (xiv) in    a heterodimeric form;-   (xvii) a protein as defined in any one of paragraphs (i) to (xiv) in    a oligomeric form;-   (xviii) a protein as defined in any one of paragraphs (i) to (xiv)    in a heteroligomeric form;

The protein of the present invention is preferably in isolated form. By“isolated” is meant a protein having undergone at least one purificationstep and this is conveniently defined, for example, by a compositioncomprising at least about 10% subject protein, preferably at least about20%, more preferably at least about 30%, still more preferably at leastabout 40-50%, even still more preferably at least about 60-70%, yet evenstill more preferably 80-90% or greater of subject protein relative toother components as determined by molecular weight, amino acid sequenceor other convenient means. The protein of the present invention may alsobe considered, in a preferred embodiment, to be biologically pure.

Without limiting the theory or mode of action of the present invention,the expression of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 is thought to relate to regulation of body weight and glucosehomeostasis. Modulation of these genes expression is thought, interalia, to regulate energy balance via effects on energy intake and alsoeffects on carbohydrate/fat metabolism. The energy intake effects arelikely to be mediated via the central nervous system but peripheraleffects on the metabolism of both carbohydrate and fat are possible. Theexpression of these genes may also be regulated by fasting and feeding,accordingly, regulating the expression and/or activity of these genes ortheir expression products could provide a mechanism for regulating bothbody weight and energy metabolism, including carbohydrate and fatmetabolism.

The identification of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 permits the generation of a range of therapeutic moleculescapable of modulating expression of AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and AGT-204 or modulating the activity of AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204. Modulators contemplated by thepresent invention includes agonists and antagonists of AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 expression. Antagonists ofAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 expressioninclude antisense molecules, ribozymes and co-suppression molecules.Agonists include molecules which increase promoter activity or whichinterfere with negative regulatory mechanisms. Antagonists of AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 include antibodies andinhibitor peptide fragments. All such molecules may first need to bemodified to enable such molecules to penetrate cell membranes.Alternatively, viral agents may be employed to introduce geneticelements to modulate expression of AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and AGT-204. In so far as AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and AGT-204 acts in association with other genes such as the obgene which encodes leptin, the therapeutic molecules may target theAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 and ob genes ortheir translation products.

The present invention contemplates, therefore, a method for modulatingexpression of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 ina mammal, said method comprising contacting the AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 gene with an effective amount of amodulator of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204expression for a time and under conditions sufficient to up-regulate ordown-regulate or otherwise modulate expression of AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204. For example, a nucleic acidmolecule encoding AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 or a derivative or homolog thereof may be introduced into a cellto enhance the ability of that cell to produce AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204, conversely, AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 antisense sequences such asoligonucleotides may be introduced to decrease the availability ofAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 molecules.

Another aspect of the present invention contemplates a method ofmodulating activity of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 in a mammal, said method comprising administering to said mammala modulating effective amount of a molecule for a time and underconditions sufficient to increase or decrease AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 activity. The molecule may be aproteinaceous molecule or a chemical entity and may also be a derivativeof AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 or itsligand.

Modulating levels of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 expression is important in the treatment of a range ofconditions such as obesity and obesity related conditions including,anorexia, energy imbalance, diabetes, metabolic syndrome, dyslipidemia,hypertension, insulin resistance and muscle development conditions. Itmay also be useful in the agricultural industry to assist in thegeneration of leaner animals, or where required, more obese animals.Accordingly, the mammal contemplated by the present invention includesbut is not limited to humans, primates, livestock animals (e.g. pigs,sheep, cows, horses, donkeys), laboratory test animals (e.g. mice, rats,guinea pigs, hamsters, rabbits), companion animals (e.g. dogs, cats) andcaptured wild animals (e.g. foxes, kangaroos, deer). A particularlypreferred host is a human, primate or livestock animal.

Accordingly, the present invention contemplates therapeutic andprophylactic uses of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/orAGT-204 amino acid and nucleic acid molecules in addition to AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204 agonistic andantagonistic agents.

The present invention contemplates, therefore, a method of modulatingexpression of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 ina mammal, said method comprising contacting the AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 genes with an effective amount ofan agent for a time and under conditions sufficient to up-regulate,down-regulate or otherwise module expression of AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and/or AGT-204. For example, antisensesequences such as oligonucleotides may be utilized.

Conversely, nucleic acid molecules encoding AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 or derivatives thereof may be introduced toup-regulate one or more specific functional activities.

Another aspect of the present invention contemplates a method ofmodulating activity of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 in a subject, said method comprising administering to saidsubject a modulating effective amount of an agent for a time and underconditions sufficient to increase or decrease AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 activity.

Modulation of said activity by the administration of an agent to amammal can be achieved by one of several techniques, including but in noway limited to introducing into said mammal a proteinaceous ornon-proteinaceous molecule which:

-   (i) modulates expression of AGT-109, AGT-407, AGT-408, AGT-409,    AGT-601 and/or AGT-204;-   (ii) functions as an antagonist of AGT-109, AGT-407, AGT-408,    AGT-409, AGT-601 and/or AGT-204;-   (iii) functions as an agonist of AGT-109, AGT-407, AGT-408, AGT-409,    AGT-601 and/or AGT-204.

Said proteinaceous molecule may be derived from natural or recombinantsources including fusion proteins or following, for example, naturalproduct screening. Said non-proteinaceous molecule may be, for example,a nucleic acid molecule or may be derived from natural sources, such asfor example natural product screening or may be chemically synthesized.The present invention contemplates chemical analogs of AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and/or AGT-204 or small molecules capable ofacting as agonists or antagonists. Chemical agonists may not necessarilybe derived from AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/orAGT-204 but may share certain conformational similarities.Alternatively, chemical agonists may be specifically designed to mimiccertain physiochemical properties. Antagonists may be any compoundcapable of blocking, inhibiting or otherwise preventing AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204 from carrying outtheir normal biological functions. Antagonists include monoclonalantibodies and antisense nucleic acids which prevent transcription ortranslation of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/orAGT-204 genes or mRNA in mammalian cells. Modulation of expression mayalso be achieved utilizing antigens, RNA, ribosomes, DNAzymes, RNAaptamers or antibodies.

Said proteinaceous or non-proteinaceous molecule may act either directlyor indirectly to modulate the expression of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and/or AGT-204 or the activity of AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and/or AGT-204. Said molecule acts directly ifit associates with AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/orAGT-204 or AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204 tomodulate expression or activity. Said molecule acts indirectly if itassociates with a molecule other than AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and/or, AGT-204 or AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and/or AGT-204 which other molecule either directly orindirectly modulates the expression or activity of AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and/or AGT-204 or AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and/or AGT-204. Accordingly, the method of the presentinvention encompasses the regulation of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and/or AGT-204 or AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and/or AGT-204 expression or activity via the induction of acascade of regulatory steps.

The molecules which may be administered to a mammal in accordance withthe present invention may also be linked to a targeting means such as amonoclonal antibody, which provides specific delivery of these moleculesto the target cells.

A further aspect of the present invention relates to the use of theinvention in relation to mammalian disease conditions. For example, thepresent invention is particularly useful but in no way limited to use ina therapeutic or prophylactic treatment of obesity, anorexia, diabetesor energy imbalance.

Accordingly, another aspect of the present invention relates to a methodof treating a mammal suffering from a condition characterized by one ormore symptoms of obesity, anorexia, diabetes and/or energy imbalance,said method comprising administering to said mammal an effective amountof an agent for a time and under conditions sufficient to modulate theexpression of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204or sufficient to modulate the activity of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and/or AGT-204.

In another aspect, the present invention relates to a method of treatinga mammal suffering from a disease condition characterized by one or moresymptoms of obesity, anorexia, diabetes or energy imbalance, said methodcomprising administering to said mammal an effective amount of AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204 or AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and/or AGT-204.

An “effective amount” means an amount necessary at least partly toattain the desired immune response, or to delay the onset or inhibitprogression or halt altogether, the onset or progression of a particularcondition of the individual to be treated, the taxonomic group of theindividual to be treated, the degree of protection desired, theformulation of the vaccine, the assessment of the medical situation, andother relevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.

In accordance with these methods, AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and/or AGT-204 or AGT-109, AGT-407, AGT-408, AGT-409, AGT-601and/or AGT-204 or agents capable of modulating the expression oractivity of said molecules may be co-administered with one or more othercompounds or other molecules. By “co-administered” is meant simultaneousadministration in the same formulation or in two different formulationsvia the same or different routes or sequential administration by thesame or different routes. By “sequential” administration is meant a timedifference of from seconds, minutes, hours or days between theadministration of the two types of molecules. These molecules may beadministered in any order.

In yet another aspect, the present invention relates to the use of anagent capable of modulating the expression of or AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and/or AGT-204 or a derivative, homolog oranalog thereof in the manufacture of a medicament for the treatment of acondition characterized by obesity, anorexia, diabetes and/or energyimbalance.

In still yet another aspect, the present invention relates to the use ofan agent capable of modulating the activity of AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and/or AGT-204 or a derivative, homolog,analog, chemical equivalent or mimetic thereof in the manufacture of amedicament for the treatment of a condition characterized by obesity,anorexia, diabetes and/or energy imbalance.

A further aspect of the present invention relates to the use of AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204 or derivative, homologor analog thereof or AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/orAGT-204 or derivative, homolog, analog, chemical equivalent or mimeticthereof in the manufacture of a medicament for the treatment of acondition characterized by obesity, anorexia, diabetes and/or energyimbalance.

Still yet another aspect of the present invention relates to agents foruse in modulating the expression of AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and/or AGT-204 or a derivative, homolog or analog thereof.

A further aspect relates to agents for use in modulating AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204 activity or aderivative, homolog, analog, chemical equivalent or mimetic thereof.

Still another aspect of the present invention relates to AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204 or derivative, homologor analog thereof or AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/orAGT-204 or derivative, homolog, analog, chemical equivalent or mimeticthereof for use in treating a condition characterized by one or moresymptoms of obesity, anorexia, diabetes and/or energy imbalance.

In a related aspect of the present invention, the mammal undergoingtreatment may be a human or an animal in need of therapeutic orprophylactic treatment.

Accordingly, the present invention contemplates in one embodiment acomposition comprising a modulator of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 expression or AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 activity and one or more pharmaceuticallyacceptable carriers and/or diluents. In another embodiment, thecomposition comprises AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 or a derivative, homolog, analog or mimetic thereof and one ormore pharmaceutically acceptable carriers and/or diluents. Thecompositions may also comprise leptin or modulations of leptin activityor ob expression.

For brevity, all such components of such a composition are referred toas “active components”.

The compositions of active components in a form suitable for injectableuse include sterile aqueous solutions (where water soluble) and sterilepowders for the extemporaneous preparation of sterile injectablesolutions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi.

The carrier can be a solvent or other medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils.

The preventions of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecomponents in the required amount in the appropriate solvent withoptionally other ingredients, as required, followed by sterilization by,for example, filter sterilization, irradiation or other convenientmeans. In the case of sterile powders for the preparation of sterileinjectable solutions, the preferred methods of preparation are vacuumdrying and the freeze-drying technique which yield a powder of theactive ingredient plus any additional desired ingredient from previouslysterile-filtered solution thereof.

When AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 andAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 includingAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 themselves aresuitably protected, they may be orally administered, for example, withan inert diluent or with an assimilable edible carrier, or it may beenclosed in hard or soft shell gelatin capsule, or it may be compressedinto tablets, or it may be incorporated directly with the food of thediet. For oral therapeutic administration, the active compound may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least1% by weight of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 5 to about 80% of the weight of the unit. The amount of activecompound in such therapeutically useful compositions is such that asuitable dosage will be obtained. Preferred compositions or preparationsaccording to the present invention are prepared so that an oral dosageunit form contains between about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such asucrose, lactose or saccharin may be added or a flavouring agent such aspeppermint, oil of wintergreen, or cherry flavouring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired as herein disclosed in detail.

The principal active component may be compounded for convenient andeffective administration in sufficient amounts with a suitablepharmaceutically acceptable carrier in dosage unit form. A unit dosageform can, for example, contain the principal active component in amountsranging from 0.5 μg to about 2000 mg. Expressed in proportions, theactive compound is generally present in from about 0.5 μg to about 2000mg/ml of carrier. In the case of compositions containing supplementaryactive ingredients, the dosages are determined by reference to the usualdose and manner of administration of the said ingredients.

In general terms, effective amounts of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 will range from 0.01 ng/kg/body weight toabove 10,000 mg/kg/body weight. Alternative amounts range from 0.1ng/kg/body weight to above 1000 mg/kg/body weight. AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 may be administered per minute,hour, day, week, month or year depending on the condition being treated.The route of administration may vary and includes intravenous,intraperitoneal, sub-cutaneous, intramuscular, intranasal, viasuppository, via infusion, via drip, orally or via other convenientmeans.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule capable of modulating AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 expression or AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 activity. The vector may, forexample, be a viral vector.

Still another aspect of the present invention is directed to antibodiesto AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 and theirderivatives and homologs. Such antibodies may be monoclonal orpolyclonal and may be selected from naturally occurring antibodies toAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 or may bespecifically raised to AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 or derivatives or homologs thereof. In the case of the latter,AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 or theirderivatives or homologs may first need to be associated with a carriermolecule. The antibodies and/or recombinant AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 or their derivatives of the presentinvention are particularly useful as therapeutic or diagnostic agents.

For example, AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 andtheir derivatives can be used to screen for naturally occurringantibodies to AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204which may occur in certain autoimmune diseases or where cell death isoccurring. These may occur, for example, in some autoimmune diseases.Alternatively, specific antibodies can be used to screen for AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204. Techniques for suchassays are well known in the art and include, for example, sandwichassays and ELISA.

Antibodies to AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 ofthe present invention may be monoclonal or polyclonal and may beselected from naturally occurring antibodies to the AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 or may be specifically raised tothe AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 or theirderivatives. In the case of the latter, the AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204 protein may need first to be associatedwith a carrier molecule. Alternatively, fragments of antibodies may beused such as Fab fragments. Furthermore, the present invention extendsto recombinant and synthetic antibodies and to antibody hybrids. A“synthetic antibody” is considered herein to include fragments andhybrids of antibodies. The antibodies of this aspect of the presentinvention are particularly useful for immunotherapy and may also be usedas a diagnostic tool or as a means for purifying AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204.

For example, specific antibodies can be used to screen for AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 proteins. The latterwould be important, for example, as a means for screening for levels ofAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 in a cellextract or other biological fluid or purifying AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 made by recombinant means fromculture supernatant fluid. Techniques for the assays contemplated hereinare known in the art and include, for example, sandwich assays andELISA.

It is within the scope of this invention to include any secondantibodies (monoclonal, polyclonal or fragments of antibodies) directedto the first mentioned antibodies discussed above. Both the first andsecond antibodies may be used in detection assays or a first antibodymay be used with a commercially available anti-immunoglobulin antibody.An antibody as contemplated herein includes any antibody specific to anyregion of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204.

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith the enzyme or protein and either type is utilizable forimmunoassays. The methods of obtaining both types of sera are well knownin the art. Polyclonal sera are less preferred but are relatively easilyprepared by injection of a suitable laboratory animal with an effectiveamount of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204, orantigenic parts thereof, collecting serum from the animal, and isolatingspecific sera by any of the known immunoadsorbent techniques. Althoughantibodies produced by this method are utilizable in virtually any typeof immunoassay, they are generally less favoured because of thepotential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art. (See, for example, Douillard and Hoffman, Basic Facts aboutHybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981;Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and Milstein,European Journal of Immunology 6: 511-519, 1976).

Another aspect of the present invention contemplates a method fordetecting AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 or aderivative or homolog thereof in a biological sample from a subject,said method comprising contacting said biological sample with anantibody specific for AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 or their antigenic derivatives or homologs for a time and underconditions sufficient for a complex to form, and then detecting saidcomplex.

The presence of the complex is indicative of the presence of AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204. This assay may bequantitated or semi-quantitated to determine a propensity to developobesity or other conditions or to monitor a therapeutic regimum.

The presence of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204may be accomplished in a number of ways such as by Western blotting andELISA procedures. A wide range of immunoassay techniques are availableas can be seen by reference to U.S. Pat. Nos. 4,016,043, 4,424,279 and4,018,653. These, of course, includes both single-site and two-site or“sandwich” assays of the non-competitive types, as well as in thetraditional competitive binding assays. These assays also include directbinding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays. Anumber of variations of the sandwich assay technique exist, and all areintended to be encompassed by the present invention. Briefly, in atypical forward assay, an unlabelled antibody is immobilized on a solidsubstrate and the sample to be tested brought into contact with thebound molecule. After a suitable period of incubation, for a period oftime sufficient to allow formation of an antibody-AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and AGT-204 complex, a second antibodyspecific to the AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204,labelled with a reporter molecule capable of producing a detectablesignal, is then added and incubated, allowing time sufficient for theformation of another complex of antibody-AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and AGT-204-labelled antibody. Any unreacted materialis washed away, and the presence of AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and AGT-204 is determined by observation of a signal produced bythe reporter molecule. The results may either be qualitative, by simpleobservation of the visible signal, or may be quantitated by comparingwith a control sample containing known amounts of hapten. Variations onthe forward assay include a simultaneous assay, in which both sample andlabelled antibody are added simultaneously to the bound antibody. Thesetechniques are well known to those skilled in the art, including anyminor variations as will be readily apparent. In accordance with thepresent invention, the sample is one which might contain AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 including cell extract,tissue biopsy or possibly serum, saliva, mucosal secretions, lymph,tissue fluid and respiratory fluid. The sample is, therefore, generallya biological sample comprising biological fluid but also extends tofermentation fluid and supernatant fluid such as from a cell culture.

The solid surface is typically glass or a polymer, the most commonlyused polymers being cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene. The solid supports may be in theform of tubes, beads, discs or microplates, or any other surfacesuitable for conducting an immunoassay. The binding processes arewell-known in the art and generally consist of cross-linking, covalentlybinding or physically adsorbing, the polymer-antibody complex to thesolid surface which is then washed in preparation for the test sample.An aliquot of the sample to be tested is then added to the solid phasecomplex and incubated for a period of time sufficient (e.g. 2-40 minutesor overnight if more convenient) and under suitable conditions (e.g.from room temperature to about 37° C.) to allow binding of any subunitpresent in the antibody. Following the incubation period, the antibodysubunit solid phase is washed and dried and incubated with a secondantibody specific for a portion of AGT-109, AGT-407, AGT-408, AGT-409,AGT-601 and AGT-204. The second antibody is linked to a reportermolecule which is used to indicate the binding of the second antibody toAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204.

An alternative method involves immobilizing the target molecules in thebiological sample and then exposing the immobilized target to specificantibody which may or may not be labelled with a reporter molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound target may be detectable by direct labellingwith the antibody. Alternatively, a second labelled antibody, specificto the first antibody is exposed to the target-first antibody complex toform a target-first antibody-second antibody tertiary complex. Thecomplex is detected by the signal emitted by the reporter molecule.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide-containing molecules (i.e.radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, β-galactosidase and alkaline phosphatase, amongst others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable colour change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labelledantibody is added to the first antibody hapten complex, allowed to bind,and then the excess reagent is washed away. A solution containing theappropriate substrate is then added to the complex ofantibody-antigen-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of hapten which was present in the sample. A“reporter molecule” also extends to use of cell agglutination orinhibition of agglutination such as red blood cells on latex beads, andthe like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody absorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic colour visually detectable with a lightmicroscope. As in the EIA, the fluorescent-labelled antibody is allowedto bind to the first antibody-hapten complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength. The fluorescence observed indicatesthe presence of the hapten of interest. Immunofluorescence and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

The present invention also contemplates genetic assays such as involvingPCR analysis to detect AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 andAGT-204 or their derivatives.

The assays of the present invention may also extend to measuringAGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 or AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and AGT-204 in association with ob orleptin.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 Partial Sequence of Psammomys obesus AGT-109

AGT-109 was identified using differential display PCR of hypothalamuscDNA from diabetic and non-diabetic Psammomys obesus.

The partial nucleotide sequence is as follows:—

-   -   GATTTTGGTTGGCAATAAATGTGACTTGGAAGATGAGCGGGTAGTTGGCAAAGAACAAGGC        CAGAATTTAGCAAGACAGTGGTGTAACTGTGCCTTTTTAGAATCTTCTGCAAAGTCAAAGA        TCAACGTTAATGAGGTCACTTTTCACAACTATGCTTATAGACTCTTATTTTAAATACCTGA        TATTTTATGATCTGGTCAGACAGATAAATAGAAAAACACCAGTG [SEQ ID NO:1]

EXAMPLE 2 AGT-109 Gene Expression

Gene expression studies using real-time PCR (RT-PCR) showed there was asignificant difference in AGT-109 gene expression in the fasted A and Banimals compared to the fed control animals (Group A p<0.001; Group Bp=0.018) but no difference in the C animals (p=0.19; FIG. 1). There wasno significant difference between Group A, B and C animals in the fed orfasted state. When data from all animals were pooled, there was asignificant increase in AGT-109 expression in fasted animals compared tofed (p<0.001; FIG. 2). There were no significant correlations betweenAGT-109 expression and insulin, body weight, body fat or glucose levels.When the experiment was repeated, hypothalamic AGT-109 expression wasnot significantly different in two-week energy restricted Group A, B orC animals, or between all controls and all restricted animals. Incontrol animals, AGT-109 expression in Group A animals was significantlyhigher than expression in Group C control animals (p=0.008), and tendedto be higher than control Group B animals (p=0.07) (FIG. 3).

In control animals, there was also a negative association betweenAGT-109 expression and percent body fat (p<0.05) and pre-insulinconcentrations (p=0.026). With all animals combined, there was asignificant negative association between AGT-109 expression andpreglucose (p=0.037) and a trend for a negative association withpreinsulin (p=0.07).

EXAMPLE 3 AGT-109 Gene Homology

Significant matches using BLAST (version 2.2.1 [Apr. 13, 2001]) withGenbank nr and dbest databases showed that AGT-109 shares 96% homologywith human RAP1A (Accession Number AL049557), a member of the rasoncogene family. AGT-109 shares similar homology to the bovine rasp21-like GTP binding protein (95%).

Ras oncogenes are ubiquitously expressed, evolutionarily-conservedmolecular switches that couple extracellular signals to various cellularresponses (Kitayama et al., Cell 56: 77-84, 1989). Ras oncogenes encodeproteins that are analagous to normal G-proteins except that an aminoacid substitution results in continuous activation of the counterfeitG-protein. The G-proteins normally bind and hydrolyze GTP, however, themutation impairs their GTPase activity and thus interferes with thenormal shut-off mechanism. RAPIA shares approximately 50% amino acididentity with the classical ras proteins (Bos et al., Nat. Rev. Mol.Cell Biol. 2(5): 369-377, 2001).

Rap1 (also known as KREV1, KREV-1 and SMGP21) is the closest relative ofRas and may regulate Ras-mediated signalling. The most strikingdifference between the RAP and ras proteins is at amino acid 61, whichis glutamine in ras and threonine in RAP protein (Kitayama et al., 1989,supra). RAP1A has been mapped to chromosome 1p13.3 and there is apseudogene (KREV1P) at 14q24.3 (Takai et al., Cytogenet. Cell Genet. 63:59-61, 1993).

RAP1A has been identified as being cytoplasmic and belonging to the rapsub-family. It is thought to be a GTPase (displaying enzymic activitythat hydrolyzes GTP to GDP and orthophosphate) and involved in cellcycle control and signal transduction pathways. Rap1 is activated byextracellular signals through several regulatory proteins. It mayfunction in diverse processes ranging from modulation of growth anddifferentiation to secretion, integrin-mediated cell adhesion andmorphogenesis (Bos et al., 2001, supra).

Several domains have been identified in the RAP1A protein, including Rasfamily, Rab subfamily of small GTPases, Ras subfamily of RAS smallGTPases, Rho (Ras homology) subfamily of Ras-like small GTPases, and Ran(Ras-related nuclear proteins)/TC4 subfamily of small GTPases.

These domains have been associated with a number of functions, includingvesicle trafficking (Woodman, Curr. Biol. 8(6): R199-210, 1998; Lazar etal., Trends Biochem. Sci. 22(12: 468-472, 1997; Novick and Zerial, Curr.Opin. Cell Biol. 9(4): 496-504, 1997; Haubruck et al., EMBO J. 6(13):4049-4053, 1987; Gallwitz et al., Nature 306(5944): 704-707, 1983),coupling receptor tyrosine kinases and G protein receptors to proteinkinase cascades (Downward, Curr. Opin. Genet. Dev. 8(1): 49-54, 1998;Lloyd, Curr. Opin. Genet. Dev. 8(1): 43-48, 1998; Wittinghofer and Pai,Trends Biochiem. Sci. 16(10): 382-387, 1991; Schlichting et al., Nature345(6273): 309-315, 1990; Pai et al., Nature 341(6239) 209-214 1989;Shih et al., Nature 287(5784): 686-691, 1980) and active transport ofproteins through nuclear pores (Richards et al., Science 276(5320):1842-1844, 1997; Yoneda, J. Biochem. 121(5): 811-817, 1997; Lounsbury etal., J. Biol. Chem. 271(51): 32834-32841, 1996; Koepp and Silver, Cell87(1): 1-4, 1996; Scheffzek et al., Nature 374(6520): 378-381, 1995;Matsumoto and Beach, Cell 66(2): 347-360, 1991).

EXAMPLE 4 Partial Sequence of Psammomys obesus AGT-407

AGT-407 was identified by Suppression Subtractive Hybridization (SSH)[also referred to as Representational Difference Analysis (RDA)] ofliver cDNA from diabetic and non-diabetic Psammomys obesus.

The partial nucleotide sequence is as follows:—

-   -   GAGGGATGNGGACAATGGCCTTTCCTTGTCATCTTTAAGTGACTGGTACAACACTTCTGTT        ATGAGAAAAGTGAAATTTTATGATGAAAACACAAGGCAGTGGTGGATGCCAGATACTGGAG        GAGCCAACATCCCAGCTCTGAATGAGCTGCTGTCTGTATGGAACATGGGGTTCAGTGACGG        CCTGTATGAAGGGGAATTTGTCCTGGCAAACCATGACATGTATTATGCGTCGGGGTGCAGC        ATCGCCAGGTTTCCAGAAGATGGTGTTGTGATCACACAGACTTTCAAGGATCAAGGATTGG        AGGTCTTAAAACAAGAGACAGCAGTTGTTGAAAATGTTCCCATTTTGGGGCTTTATCAGAT        TCCAGCTGAAGGTGGAGGTCGTATTGTGCTGTATGGAGACTTCAACTGCTTGGATGACAGT        CACAGACAGAAGGACTGNTTTTGGCTTCTGGATGCGCTCCTTNAGTACCTCGG [SEQ ID        NO:2]

EXAMPLE 5 AGT-407 Gene Expression

AGT-407 was not normally distributed. Non-parametric (Kruskal-Wallis)tests indicated a significant difference between Groups (p=0.036). UsingMann-Whitney, tests found Group A fasted animals had significantlyhigher gene expression than C fed (p=0.014) and B fed (p=0.029; FIG. 4).No other differences were found between Groups.

Fasted animals had significantly higher AGT-407 expression (p=0.003)compared to fed animals using Mann-Whitney (FIG. 5).

EXAMPLE 6 AGT-407 Gene Homology

AGT-407 showed strong nucleotide homology to mouse site-1 protease,mouse and rat subtilisin/kexin isozyme SKI-1 precursor and humanmembrane-bound transcription factor protease, site 1 and KIAA0091 gene(BLASTN version 2.2.1 [Apr. 13, 2001]).

Site-1 protease is the same gene as SKI-1 and KIAA0091 and is also knownas membrane-bound transcription factor protease, site 1; site-1 protease(subtilisin-like, sterol-regulated, cleaves sterol regulatory elementbinding proteins) and subtilisin/kexin isozyme-1 preproprotein.

Site-1 protease (S1P) is a subtilisin-related protease that cleavessterol regulatory element-binding proteins (SREBPs) in the endoplasmicrecticulum lumen to initiate the release from membranes oftranscriptionally active amino-terminal fragments of SREBPs. A secondprotease (Site-2 protease) is also involved in this process but onlyafter site-1 protease has acted.

SREBPs are membrane-embedded proteins, requiring proteolytic release ofthe active portions which move to the nucleus. SREBPs aretranscription-regulating proteins that form a feedback system to adjustthe expression of genes encoding the LDL receptor and multiple enzymesin the cholesterol and fatty acid biosynthetic pathways.

Within the nucleus, SREBPs activate transcription of genes involved inthe cholesterol biosynthesis pathway (regulating genes such as HMG CoAsynthase, HMG CoA reductase, farnesyl diphosphate synthase, squalenesynthase, and the LDL receptor) and fatty acid biosynthesis (AcetylCoAcarboxylase (ACC), fatty acid synthase (FAS), stearoylCoA desaturase-1(SCD)).

Cells that lack mature SREBPs have near-complete block of cholesterolsynthesis and LDL receptor activity and rates of fatty acid synthesisreduced by 50%. Animals that over-express the SREBP1a isoformoverproduce cholesterol and fatty acids and have increased liver sizedue to increased triglycerides (TG) and cholesterol esters. However,plasma cholesterol and TG are reduced, possibly due to increased LDLreceptor activity in the liver. Kim et al. 1998 have shown that leptinappears to be an SREBP-responsive gene.

Human site-1 protease is located on chromosome 16 and has been mapped tothe interval 16q24. This gene is more than 60 kb long and contains 23exons and 22 introns. Its transcription-initiation site within exon 1 isseparate from the initiation codon in exon 2. Analysis of theexon/introns structure revealed that the SIP gene consists of a mosaicof functional units: exon 1 encodes the 5′ non-translated region; exon 2encodes the amino-teminal signal sequence; and exons 2 and 3 encode thepre-peptide sequence that is released when SIP is self-activated byintramolecular cleavage. Exons 5-10 encode the subtilisin-homologydomain necessary for catalytic activity, and exon 23 encodes thetransmembrane region. (Nakajima et al., 2000, supra). FIG. 6 depicts thegenomic structure of the human S1P gene.

The putative promoter region had a highly G/C-rich region containing abinding site for ADD1/SREBP-1 as well as Sp1 and AP2 sites. Therefore,expression of the S1P gene may be under the control of SREBP-1, a keyregulator of the expression of genes essential for intracellular lipidmetabolism.

Shown in FIG. 7 is the relationship between exon organization andfunctional domains of SIP. A translation initiation codon (ATG) ispresent in exon 2 and a translation stop codon (TGA) is present in exon23. Upward arrows indicate SIP processing site; SS, signal sequence; TM,transmembrane domain.

EXAMPLE 7 Partial Sequence of Psammomys obesus AGT-408

AGT-408 was identified by SSH (RDA) of liver cDNA from diabetic andnon-diabetic Psammomys obesus.

The partial nucleotide sequence is as follows:—

-   -   CCGCCCGGGCAGGACTTGAGNCCACCCCTGTAGATCTGGCTTCTATTTCTCCAGCTATTGC        NGTCCTCAAGTAAAGGTCTGCAGCTAGCAGGCAGGTGTAAACCAGCCATTAAGTCTTGGCA        GATACCNCACTGTGGGTGTTAGATCTAGATCATTAAAATATTGGTAAAAAGTGATCTATCA        TGAGATTAAGCTTCCTAAAGAAGAAAGTAGCTATATANCAAGAGTCTATTAGAAGAAAGTA        GAGGAGCTGCTGAGTAAAAATCCAGCTGTATTAAGGCAAGGAACTGGAATATTGCAAAAGG        ATACACCTCCATCTCTGAGTTTGTTTTAGATGGAAAAAGTGGAGTGGGAGTGGAAAGCTCT        TTAAGGTCAGATCTTTGATAGATGATGCTCTGCATAGACATTGGTGCTGTAGAACTTAATC        AAATTGGAGCATGCATGGGCATTACCTGGGGTTCTCGTTAAACTTCTTTGTTATCATGAAA        TTCTGGGCTGGGACACAAAGGAAGCATTTGAGAAAGCTCTGCTGCGNCTAATGCCACTTTG        AGTTGTAAGAACCTCCTAGAATGTCAGGAGGACAAGGTGCCAGAAGCATATGCACTAANCT        CAATATGAAGATAAGGTANGGGACTANAAAGGGATTCANAT [SEQ ID NO:3]

EXAMPLE 8 AGT-408 Gene Expression

AGT-408 was normally distributed. One way ANOVA with an LSD post hoctest found Group A fed animals had significantly lower gene expressionthan fasted Group A animals (p=0.002) and fed Group B animals (p=0.041,FIG. 8). There was no significant difference when all animals werecombined (FIG. 9).

EXAMPLE 9 AGT-408 Gene Homology

The AGT-408 sequence did not show significant homology with anything onthe public database (BLASTN version 2.2.1 [Apr. 13, 2001]).

EXAMPLE 10 Partial Sequence of Psammomys obesus AGT-409

AGT-409 was identified by SSH (also referred to as RDA) of liver cDNAfrom diabetic and non-diabetic Psammomys obesus.

The partial nucleotide sequence is as follows:—

-   -   CCTCACACCAGTTCTTTTCTTCATAATGGACCGGATATAAAGCTTCTTGGCATCCCAGAAC        TTTGGCATACAGCTCACAGATTTTCTTCTTCCTCATTTCTTTTTGTAGCTTAGCAAGTCGA        TCTGCTTTCCGGGCAAGTATGAAGCCCTTGATGGCAGGAAATGATCCATCTGGTTTGGTAT        CATCCAAAGTGATTGAAATTGGAGCTTCCTCATCTTCAATTAGCATGCAGCCACAATAGTC        CTTTTTCTTCCAGAAGGCTTCCTTGTAATACACCATGCACTTTATTACAGCACCCATTGGT        AGACGCTGAATTAACTGGTTTCTCTCAGATGGAAGCTCTGGTTTAAAGTGGATCTTGGTAG        TCAAAGCTGGTGGGATGGCACTAATTACGTATTTGCACTCATAGTGGTCATGATTCAGTGT        CTCTACAATGAT [SEQ ID NO:4]

EXAMPLE 11 AGT-409 Gene Expression

AGT-409 was normally distributed. One way ANOVA with a Games Howell posthoc test found Group A fed animals had significantly higher geneexpression than fasted Group A (p=0.048), B (p=0.029) and C (p=0.024)animals (FIG. 10). An independent samples t-test showed fed animals hadsignificantly higher gene expression than fasted animals (p<0.001, FIG.11).

EXAMPLE 12 AGT-409 Gene Homology

AGT-409 showed strong nucleotide homology to the rat and human monoamineoxidase A (MAOA) gene (BLASTN version 2.2.1 [Apr. 13, 2001]). MAOA isalso known as amine oxidase (flavin containing). The human MAOA gene islocated on chromosome X and has been mapped to the interval Xp11.4-11.3.

There are two monoamine oxidase isoforms, designated A and B, encoded byseparate genes (Kochersperger et al., J. Neurosci. Res. 16: 601-616,1986; Lan et al., Genomics 4: 552-559, 1989). MAOA and MAOB are 70%homologous at the amino acid level. Both enzymes are located in theouter mitochondrial membrane where they catalyse the oxidativedeamination of biogenic amines (Brunner et al., Science 262: 578-580,1993a). They are found in most cell types including liver and brain(Schnaitman et al., J. Cell Biol 32(3): 719-735, 1967).

MAOA knockout studies in mice showed that serotonin concentrations wereincreased up to 9-fold, and serotonin-like immunoreactivity was presentin catecholaminergic neurons in pup brains. In pup and adult brains,norepinephrine concentrations were increased up to 2-fold andcytoarchitectural changes were observed in the somatosensory cortex. Pupbehavioural alterations, including trembling, difficulty in righting andfearfulness, were reversed by the serotonin synthesis inhibitorparachlorophenylalanine. Adults manifested a distinct behaviouralsyndrome, including enhanced aggression in males (Cases et al., Science268: 1763-1766, 1995). Similar results were demonstrated by Shih et al.(Annu. Rev. Neurosci. 22: 197-217, 1999). Obesity and diabetes relatedphenotypes were not examined in these MAOA knockout studies.

MAOA has been localized to chromosome Xp11.4-p11.23. Sims et al.(Neurons 2: 1069-1076, 1989) demonstrated that patients with Norriedisease possess a submicroscopic deletion in the region of Xp21-p11,resulting in the absence of MAOA gene. Some of the features of Norriedisease, including mental retardation, autistic behaviour, abnormalsexual maturation, peripheral autonomic dysfunction, motorhyperactivity, seizures and sleep disturbance, are likely to be due tomutation in the MAOA or MAOB genes. Obesity and diabetes relatedphenotypes were not examined in these patients.

Linkage analysis with a form of X-linked nondysmorphic mild mentalretardation demonstrated a maximal multipoint lod score of 3.69 forlinkage to MAOA at Xp11.4-p11.23 (Brunner et al., 1993a, supra). Allaffected males showed characteristic abnormal behaviour, in particularaggression and sometimes violence. Other types of impulsive behaviourincluded arson, attempted rape and exhibitionism. Attempted suicide wasreported in a single case. Results of urinalysis in three affected malesindicated a marked disturbance of monoamine metabolism. Platelet MAOBactivity was normal. In a later publication, Brunner et al. (Am. J. Hum.Genet. 52: 1032-1039, 1993b) reported that each of five affected maleshad a point mutation in the eighth exon of the MAOA structural gene,which changed a glutamine to a stop codon.

MAOA inhibitors are effective in the treatment of panic disorder. Anassociation study with a repeat polymorphism in the promoter of the MAOAgene has been significantly associated with panic disorder (Deckert etal., Hum. Molec. Genet. 8: 621-624, 1999). Some studies have found asignificant association between MAOA polymorphisms and bipolar affectivedisorder (Lim et al., (Letter) Am. J. Hum. Genet. 54: 1122-1124, 1994;Kawada et al., (Letter) Am. J. Hum. Genet. 56: 335-336, 1995), whereasothers have not (Nothen et al., (Letter) Am. J. Hunt. Genet. 57:975-977,1995).

In vitro studies have demonstrated MAOA activity can vary over 50-foldin control subjects (Breakefield et al., Psychiatry Res. 2(3): 307-314,1980). Increased MAOA activity occurs with ageing and glucocortocoidtreatment (Edelstein and Breakefield, Cell Mol. Neurobiol. 6(2):121-150, 1986). Hotamnisligil and Breakefield (Am. J. Hum. Genet. 49:383-392, 1991) determined the coding sequence of mRNA for MAOA. Usingtwo RFLPs plus another located in the non-coding region of the MAOAgene, they found statistically significant associations betweenparticular alleles and the level of MAO activity in human malefibroblast lines. They interpreted this to indicate that the MAOA geneis itself a major determinant of activity levels, apparently in partthrough non-coding, regulatory elements.

EXAMPLE 13 Partial Sequence of Psammomys obesus AGT-601

AGT-601 was discovered in silico.

The partial sequence is as follows:—

-   -   ATGGCTAACAGGGGCCCGAGCTATGGTTTAAGCCGCGAGGTGCAGGAGAAGATCGAGCAG        AAGTATGACGCGGACCTGGAGAACAAGCTGGTGGACTGGATCATCCTACAGTGTGCCGAG        GACATAGAGCACCCGCCCCCGGGCAGGGCCCATTTTCAGAAATGGTTGATGGACGGGACG        GTCCTGTGCAAGCTGATAAACAGTTTATACCCACCAGGACAAGAACCCATCCCCAAGATC        TCAGAGTCAAAGATGGCTTTTAAGCAGATGGAGCAGATCTCTCAGTTCCTGAAAGCAGCC        GAGGTCTATGGTGTCAGGACCACTGACATCTTTCAAACAGTGGATCTGTGGGAAGGGAAG        GACATGGCAGCTGTTCAGAGGACTCTGATGGCTCTAGGCAGTGTTGCTGTTACCAAGGAT        GATGGCTGCTACAGGGGAGAGCCATCCTGGTTTCACAGGAAAGCCCAGCAGAATCGGAGA        GGATTTTCAGAGGAGCAGCTTCGCCAGGGACAAAACGTCATAGGCCTGCAGATGGGTAGC        AACAAGGGTGCATCCCAGGCAGGCATGACGGGGTATGGGATGCCCCGGCAGATCATGTAA        [SEQ ID NO:5]

EXAMPLE 14

AGT-601 Gene Expression

FIG. 12 shows that C fed animals were significantly different to A and Bfed animals (p=0.004, p=0.005 respectively), as well as beingsignificantly different to A, B and C fasted animals (p<0.001, p=0.007,p=0.001 respectively). FIG. 13 shows that a significant difference isseen in AGT-601AGT-601 gene expression in the hypothalamus between allfed and fasted animals (p=0.015). AGT-601 gene expression in fed animalsis positively correlated with log glucose levels (p=0.027, FIG. 14) andpercent body fat (p=0.040, FIG. 15).

A significant difference was observed in AGT-601 gene expression in thehypothalamus between saline treated, 3 μg Beacon (PCT/AU98/00902 [WO99/23217] treated, 30 μg Beacon treated, and NPY and Beacon treatedgroups (p=0.015, FIG. 16). Saline treated animals were significantlydifferent to NPY and Beacon treated animals (p=0.028), and NPY andBeacon treated animals were significantly different to 3 μg Beacontreated and 30 μg Beacon treated animals (p=0.004, p=0.005,respectively). This indicates that ICV administration of NPY and Beaconincreases the level of AGT-601 gene expression in the hypothalamus.

A significant difference was observed in AGT-601 gene expression in GT17cells between all insulin-treated groups (p=0.029) (FIG. 1). The 0 nMinsulin treatment group was significantly different to the 1 nM, 10 nM,100 nM, and 1000 nM treatment groups (p=0.005, p=0.016, p=0.006, andp=0.006, respectively). Overall, insulin treatment lead to a decrease inAGT-601 gene expression in GT17 cells.

No significant difference was observed in AGT-601 gene expressionbetween the differing glucose treatment groups. This indicates thatglucose does not have an effect on AGT-601 gene expression in GT17cells.

EXAMPLE 15 AGT-601 Gene Homology

Psammomys obesus AGT-601 nucleotide sequence has strong homology tomouse, rat and human AGT-601 at both the nucleotide (BLASTN version2.2.1 [Apr. 13, 2001]) and amino acid level.

AGT-601 is a neuronal specific protein of 206 amino acids, which hasbeen identified in rats (Ren et al., Molecular Brain Research 22:173-185, 1994). Currently there is little published about AGT-601 andits function is unknown. AGT-601 was initially detected in the brain ofrats by Western blot analysis. It was not found in the liver, kidneys,testis or heart (Ren et al., 1994, supra). The protein is widely andspecifically distributed within the rat brain, indicating that it mayhave an essential and highly-differentiated function (Ren et al., 1994,supra). Intense staining of the central nucleus and the stria terminalisindicated high levels of AGT-601 in the amygdaloid complex. This regionof the brain is thought to control a number of endocrine responses andto regulate complex behavioural functions (Ren et al., 1994, supra).

There is a high degree of sequence homology among AGT-601, calponin andSM22α. Calponin is a troponin-like molecule, present in most vertebratesmooth muscles where it binds to actin, tropomyosin, and calmodulin(Takahashi et al., Biochem. Biophys. Res. Commun. 141: 20-26, 1986;Takahashi et al., Hypertension 11: 620-626, 1988). It also interactswith brain microtubules in a calcium-independent manner through itsbinding to tubulin, indicating a potential role as a regulator in theinteraction between microfilaments and microtubules (Fujii et al.,Journal of Biochemistry 125: 869-875, 1999). SM22α, also termedtransgelin, is a globular protein expressed predominantly in smoothmuscle-containing tissues (Camoretti-Mercado et al., Genomics 49:452-457, 1998). The name transgelin reflects the transformation andshape change-sensitive actin-gelling function of the protein (Lawson etal., Cell Motil. Cytoskeleton 38: 250-257, 1997). The sequence homologybetween these proteins and AGT-601 points to a possible interaction ofAGT-601 with the cytoskeleton in neuronal cells.

AGT-601 is also highly homologous (>96%) to a novel protein found inhumans, hNP22 (Depaz et al., In: Proceedings of the AustralianNeuroscience Society: 21^(st) Annual Meeting; 2001; Brisbane ConventionCentre, Australian Neuroscience Society Incorporated, p. 191, 2001). The3′ region of the hNP22 sequence perfectly aligns with the 1005 base pairsequence of human AGT-601 mRNA listed with GENBANK (Accession NumberAF112201) (Fan et al., Journal of Neurochemistry 76: 1276-1281, 2001).There is an important exception, however, with the human AGT-601sequence containing an additional thymidine in what would have been thestop codon, resulting in a larger protein. Fan et al. (2001, supra),therefore, suggested the name hNP22 to reflect the size and homology ofthe human gene product. Recent studies on brains from human alcoholicshave revealed elevated expression of the novel gene hNP22, suggesting apossible role in alcohol dependence (Fan et al., 2001, supra). Due tohNP22 being a cytoplasmic, putative calcium-binding protein, which mayinteract with the cytoskeleton, it has been suggested that the increasedexpression observed after chronic alcohol exposure may reflect anadaptive change.

Studies on alcohol dependence in rats revealed an increase in AGT-601expression in response to alcohol withdrawal (Depaz et al., 2001,supra), suggesting a role for AGT-601 in the addiction of rats toalcohol. Due to its sequence homology with hNP22, calponin, and SM2(x,AGT-601 may also interact with the cytoskeleton and be involved with anadaptive response to chronic alcohol exposure. As previously discussed,the reward system has been implicated in addictive, compulsivebehaviours such as alcohol dependence. Therefore, AGT-601 may have arole to play in this complex system. Due to the reward system beingimplicated in the regulation of energy homeostasis and the developmentof obesity, it is plausible that AGT-601 may have a role in regulatingenergy balance.

EXAMPLE 16 Partial Sequence of Psammomys obesus AGT-204

Untranslated Region of an Unknown Protein (AGT-204) was identified asdifferentially expressed between diabetic and non-diabetic Psammomysobesus using macroarray analysis in the pancreas.

The partial sequence is as follows:—

-   -   TGACCAATAGCTTATGAAATTTAGAAGCTTTCTAATACTCGTTTTATAAATTTAATCATT        TGCTAATGGGAATTTTACCACCTNGCATTTCTGTTACAAATCTCGGCTCCAGGGAGCAAC        GCTACAACGCTACAATTCTGGAGTTGCTTTTCTTGCCTGTCACAGGAGGTCCCTGCTCGG        CAATGACCTTTGTGAGTTAGGATAATGACTTTTCTTCTTTTCTTTCTTTTTTCCTTTTGT        ACTTCAGATGTAGGAAAAAAGGATTCTGTTTCCATGTGAAAGGAACTGTAAGCTTTTAT [SEQ        ID NO:6]

EXAMPLE 17 AGT-204 Gene Expression

There was a significant difference in AGT-204 gene expression betweenthe fed and fasted A animals but not in B or C animals (A animalsp=0.017, FIG. 19). When all animals were pooled, there was a significantincrease in AGT-204 expression in fed animals compared to fasted(p=0.001, FIG. 20). There was no significant difference between A, B orC animals in either the fed or fasted state. There were no significantcorrelations between AGT-204 expression and, body weight or insulin orglucose levels.

Across the three animal groups, AGT-204 hypothalamic expression wasincreased with fasting in Group B animals (p=0.03) and tended to beincreased in Group A animals (p=0.05), (FIG. 21). Hypothalamicexpression of AGT-204 was significantly higher in animals fastedovernight compared to fed control animals (p=0.009, FIG. 22). There wasno difference in gene expression between Group A, B and C fed animals.

Hypothalamic AGT-204 expression was not significantly different inenergy restricted Group A, B or C animals, or between all controls andall restricted animals (FIG. 23). AGT-204 expression in Group A controlanimals was significantly lower than expression in Group B and C controlanimals (p<0.03). There were no associations between AGT-204 expressionand body weight, glucose and insulin in all animals or energy restrictedanimals, however, there was a relationship between body weight andAGT-204 expression in control animals only (p=0.001), (FIG. 24).

EXAMPLE 18 AGT-204 Gene Homology

The AGT-204 nucleotide sequence aligns to the untranslated region of twodifferent genes, the 3′UTR of MAP1B (microtubule associated protein 1B)and the 5′UTR of EGF repeat transmembrane also known as DEAD/H(Asp-Glu-Ala-Asp/His) box polypeptide [Alternate Symbols: DICE1,DKFZP434B105, HDB, NOTCHL2, DBI-1 and Notch2-like] (BLASTN version 2.2.1[Apr. 13, 2001]).

A paper published in 1999 by Meixner et al. (Biochemica et BiophysicaActa. 1445: 345-350, 1990) examined the apparent overlap between the 3′region of MAP1B gene with the 5′ region of Notch2-like (DBI-1) gene.They present a very convincing argument that the published structure ofthe DBI-1 cDNA is incorrect. This suggests AGT-204 is actually the mouseMAP1B gene (also known as Mtap-5).

Microtubule Associated Protein (MAP)1B was originally isolated becauseof its cross-reactivity with a polyclonal antiserum directed against theC-terminal domain of dystrophin (Lien et al., Proc. Natl. Acad. Sci. USA88: 7873-7876, 1991). A cDNA clone was isolated by Lien et al. (1991,supra) and the gene was mapped by in situ hybridization to 5q13, in veryclose proximity to the spinal muscular atrophy (SMA) locus. The SMAs area clinically heterogeneous group of neurodegenerative disorders andcomprise the second most common fatal autosomal recessive disease aftercystic fibrosis (Swash and Schwartz, Neuromyuscular Diseases (Springer,London), 2^(nd) Ed., pp. 85-112, 1988). The disease primarily affectsthe α motor neuron with secondary atrophy of skeletal muscles.

The two forms of SMA, type I and type II, have been mapped to chromosome5q (5q13) and Lien et al. (1991, supra) investigated the possibilitythat defects in MAP1B result in SMA. The maximum lod score between SMAand MAP B for combined sexes was 20.24 at a recombination fraction of0.02. The 2 recombinants between MAP1B and SMA might appear to eliminatethe possibility of an etiologic relationship between MAP1B and SMA.However, there is likely to be non-allelic heterogeneity, particularlyamong chronic cases of SMA. If MAP1B were indeed the SMA locus, it wouldbe expected to be recombinant in families that have mutations at anotherlocus. MAP1B was found to be the closest marker distal to the locus forSMA and its 5-prime end was oriented toward the centromere (Wirth etal., Genomics 15: 113-118, 1993). Although the relationship betweenMAP1B and SMA could not be conclusively determined, if MAP1B is not thegene associated with SMA it is nevertheless an extremely tightly-linkedmarker based on genetic and physical evidence (Lien et al., 1991,supra).

MAP1B is also thought to be associated with SMA because ofimmunohistochemical data for MAP1B in adult rat spinal cord(Sato-Yoshitake et al., Neuron 3(2): 229-238, 1989) and in embryonicavian spinal cord (Tucker et al., J. Comp. Neurol. 271(1): 44-55, 1988)showing intense and specific staining of motor neurons in the anteriorhorn. This finding correlates with the specific degeneration of anteriorhorn motor neurons in SMA patients (Lien et al, 1991, supra).

MAP1B is an abundant high molecular weight neuronal protein and is thefirst MAP expressed during nervous system development. It is also knownto be highly enriched in growing axons of the developing and maturenervous system (Bloom et al., Proc. Natl. Acad. Sci. USA 82(16):5404-5408, 1985; Calvert and Anderton, EMBO J. 4(5): 1171-1176, 1985;Calvert et al., Neuroscience 23(1): 131-141, 1987; Riederer et al., J.Neurocytol. 15(6): 763-775, 1986; Schoenfeld et al., J. Neurosci. 9(5):1712-1730, 1989; Tucker et al., 1988, supra), suggesting a specific rolein the initial formation and remodeling of the axonal cytoskeleton(Hammarback et al., Neuron 7: 129-139, 1991).

MAP1B is highly elongated (190 nm in length) with a small globulardomain at one end (Sato-Yoshitake et al., 1989, supra). It is a complexof one heavy chain (>200 kd) and two light chains (light chain 1 (LC1),-34 kd; light chain 3, ˜19 kd). Although there is similarity in thesubunit composition between MAP-1A and MAP1B, the heavy chains of thetwo proteins are immunologically and biochemically distinct (Bloom etal., 1985, supra, Reinderer et al., 1986, supra). Hammarback et al.(1991, supra) found that LC1 is encoded within the 3′ end of the MAP1Bheavy chain gene. Their data suggested that the heavy chain and lightchain 1 are produced by proteolytic processing of a precursorpolypeptide. This generates a novel multi-subunit microtubule-bindingdomain near the heavy chain N-terminus.

Noble et al. (J. Cell Biol. 109(6): 3367-3376, 1989) found that theMAP1B gene encoded a protein with a predicted molecular mass ofapproximately 255 kd and showed that the basic regions within theprotein containing KKEE and KKEVI motifs were responsible for theinteraction between MAP1B and microtubules in vivo (Noble et al., 1989,supra). Further, the region showns no sequence relationship to themicrotubule binding domains of kinesin, MAP2 or tau. Lien et al. (1994)completely cloned and sequenced the human MAP1B gene. The expressedprotein showed 91% overall identity with rat and mouse MAP1B and has 7exons. The third exon contains sequence not represented in mouse or ratMAP1B and is present at the 5′ end of an alternative transcript that isexpressed at approximately one-tenth the level of the full-lengthtranscript.

Neuronal microtubules are considered to have a role in dendrite and axonformation. Different portions of the developing and adult brainmicrotubules interact with different microtubule-associated proteins.MAP1B is expressed in different portions of the brain and may have arole in neuronal plasticity and brain development.

Edelmann et al. (Proc. Natl. Acad. Sci. USA 93(3): 1270-1275, 1996)generated mice with an insertion in MAP1B by gene-targeting methods.Mice homozygous for the modification died during embryogenesis while theheterozygotes exhibited a spectrum of phenotypes including slower growthrates, lack of visual acuity in one or both eyes and motor systemabnormalities. Histochemical analysis of the severely affected micedemonstrated that their Purkinje cell dendritic processes were abnormal,did not react with MAP 1B antibodies and showed reduced staining withMAP1A antibodies. Similar histologic and immunochemical changes wereobserved in the olfactory bulb, hippocampus and retina, providing abasis for the observed phenotypes.

EXAMPLE 19 Primers

Primer and probe sequences for amplification and analysis of each gene(shown in the 5′ to 3′ direction).

SYBR Green Analysis

-   AGT-109 Forward: ttggcaataaatgtgacttggaa [SEQ ID NO:7]-   AGT-109 Reverse: cgttgatctttgactttgcagaag [SEQ ID NO: 8]-   AGT-407 Forward: ggatcaaggattggaggtcttaaa [SEQ ID NO:9]-   AGT-407 Reverse: tggaatctgataaagccccaaa [SEQ ID NO:10]-   AGT-408 Forward: cacctccatctctgagtttgttttag [SEQ ID NO:11]-   AGT-408 Reverse: catcatctatcaaagatctgaccttaaag [SEQ ID NO:12]-   AGT-409 Forward: tgctttccgggcaagtatg [SEQ ID NO:13]-   AGT-409 Reverse: aatttcaatcactttggatgatacca [SEQ ID NO:14]-   AGT-204 Forward: gagttgcttttcttgcctgtca [SEQ ID NO:15]-   AGT-204 Reverse: aaagaagaaaagtcattatcctaactcaca [SEQ ID NO:16]    Taqman Analysis-   AGT-601 Forward: cgggcagggcccatt [SEQ ID NO:17]-   AGT-601 Reverse: ggtataaactgtttatcagcttgcaca [SEQ ID NO:18]-   PROBE: FAM-agaaatggttgatggacgggacggt-TAMRA [SEQ ID NO:19]-   Beta-actin Forward: gcaaagacctgtatgccaacac [SEQ ID NO:20]-   Beta-actin Reverse: gccagagcagtgatctctttctg [SEQ ID NO:21]-   Probe: FAM-tccggtccacaatgcctgggaacat-TAMRA [SEQ ID NO:22]-   Cyclophilin Forward: cccaccgtgttcttcgaca [SEQ ID NO:23]-   Cyclophilin Reverse: ccagtgctcagagcacgaaa [SEQ ID NO:24]-   Probe: FAM-cgcgtctccttcgagctgtttgc-TAMRA [SEQ ID NO:25]

EXAMPLE 25 Suppression Subtractive Hybridization (SSH)

SSH was used for gene discovery in the liver of lean Group A animals(n=3) and obese/diabetic Group C animals (n=3). Forward and reversesubtractions were performed to identify novel genes up-regulated in eachof the respective populations.

The forward subtraction to identify genes up-regulated in Group Aanimals is described below. Groups A and C were designated tester anddriver, respectively. The PCR-Select cDNA subtraction kit (Clontech,Palo Alto, USA) was used for the SSH. Experiments were conductedaccording to the manufacturer's protocol (Clontech, Palo Alto, USA) andare briefly described below.

First strand cDNA was synthesized from 0.4 μg of tester mRNA and 0.4 μgof driver mRNA in a reaction containing 20 units of AMV reversetranscriptase and a cDNA synthesis primer. The reaction was incubated at42° C. for 90 minutes. Second strand tester and driver cDNA wassynthesized in a reaction containing 24 units of DNA polymerase I, 1unit of RNase H and 4.8 units of DNA ligase. The reaction was incubatedat 16° C. for 3 hours. 6 units of T4 DNA polymerase were added andincubation continued for a further 30 minutes.

Tester and driver cDNA were digested for 90 minutes at 37° C. with 15units of the restriction endonuclease Rsa I.

The tester cDNA was divided into two equal aliquots, designated tester 1and 2. Adaptor oligonucleotide adaptor 1 was ligated to tester 1 andadaptor 2R was ligated to tester 2. The reaction containing 400 units ofT4 DNA ligase was incubated at 16° C. for 16 hours.

Following the adaptor ligation two hybridisation and two amplificationstages were performed (FIG. 26).

The first hybridization involved adding an excess of driver cDNA totester 1 and 2. The samples were denatured at 98° C. for 90 seconds, andallowed to anneal at 68° C. for 8 hours. Four different types ofmolecules (designated a, b, c, and d) were produced. Single-strandedcDNA that was common to both tester and driver annealed to form type cmolecules. Single strand cDNA that was up-regulated in the tester eitherreannealed with the complimentary tester sequence (type b molecules), orremained single stranded (type a molecules). Excess added driver ensuredthat most up-regulated cDNA remained single-stranded (a). Single anddouble-stranded driver molecules also remained (type d molecules).

The second hybridization involved denaturing another aliquot of drivercDNA at 98° C. for 90 seconds and combining this with both testers 1 and2. The reaction was allowed to anneal at 68° C. for 20 hours. Type a, b,c, and d molecules remained as well as type e molecules which werehybrids of type a molecules from tester 1 and tester 2. One cDNA strandof these molecules was ligated to adaptor 1 and the other was ligated toadaptor 2R. PCR was used to amplify these molecules.

Before amplification, a 5-minute extension phase at 75° C. “filled in”the complementary strand for each adaptor. The primer sequence wascomplimentary to the “filled in” sections on both adaptor 1 and adaptor2R. Type d molecules were not amplified because they did not have aprimer binding site. Type a and c molecules were amplified linearly asthey only had one primer binding site. Type b molecules could not beexponentially amplified as they form a pan-like structure (Diatchenko etal., Proc. Natl. Acad. Sci. USA 93(12): 6025-6030, 1996; Diatchenko etal., In: RT-PCR Methods for Gene Cloning and Analysis, Eds. Siebert, P.and Larrick, J. (Biotechniques Books, MA), pp. 213-239, 1998). Type emolecules amplified exponentially.

A second PCR was performed using two primers complimentary to the“filled in” sections of adaptors 1 and 2R, respectively. Type emolecules were further amplified. These molecules represented genesputatively up-regulated in Group A animals.

Differential Screening

Putatively up-regulated genes were screened and isolated using thePCR-Select Differential Screening Kit (Clontech, Palo Alto, USA).Screening experiments were conducted with the products of the forwardand reverse subtractions as outlined in the protocol (Clontech, PaloAlto, USA). The screening experiment with the forward subtraction isbriefly described below.

The subtracted cDNA from the SSH experiment was cloned using a T/Acloning system (TOPO TA Cloning Kit, Invitrogen, Carlsbad, USA) asdescribed in the Invitrogen protocol. The PCR products were ligated intoa pCR2.1-TOPO plasmid vector and chemically transformed into TOP10 E.coli cells. Cells were grown overnight at 37° C. on Luria-Bertani (LB)plates. White colonies, representing successfully transfected clones,were selected and grown overnight at 37° C. in LB medium. These cloneswere amplified by PCR using primers complementary to adaptors 1 and 2R.These PCR products were used to prepare cDNA dot blots.

Four identical nylon membranes were prepared for cDNA dot blots, asdescribed in the PCR Select Differential Screening Kit protocol(Clontech, Palo Alto, USA). The PCR products representing the positiveclones were cross-linked to nylon membranes using a UV Stratalinker at120 mJ (Stratagene, Austin, USA). The membranes were washed inExpressHyb (Clontech, Palo Alto, USA), a prehybridization solution.

The subtracted cDNA from the forward and reverse subtractions were usedto prepare forward and reverse probes, respectively. In additionunsubtracted cDNA from the forward and reverse experiments were used toprepare unsubtracted probes. cDNA was denatured at 95° C. for 8 minutes,and incubated at 37° C. for 30 minutes in a reaction containing α³³Plabeled DATP (50 μCi) (Geneworks, Adelaide, Australia) and 3 units ofKlenow enzyme (Clontech, Palo Alto, USA). The forward and reverse probeswere hybridized to the nylon membrane for 16 hours at 72° C. Membraneswere washed with low and high stringency wash solutions and exposed to aphosphorus plate (Molecular Dynamics, Sunnyvale, USA) for five days. Aphosphorimager (Molecular Dynamics, Sunnyvale, USA) was used to examinethe image transferred to this plate.

A clone was identified as up-regulated in Group A animals when a signalwas detected from the forward subtracted probe without a signal from thereverse subtracted probe and a more intense signal was detected from theforward unsubtracted probe than the reverse unsubtracted probe.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

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1. An isolated nucleic acid molecule comprising a sequence ofnucleotides encoding or complementary to a sequence encoding anexpression protein or a derivative or homolog thereof wherein saidnucleic acid molecule is differentially expressed in hypothalamus, liverand/or pancreatic tissue in obese animals compared to lean animals or infasted animals compared to fed animals or in diabetic animals comparedto non-diabetic animals, wherein the nucleic acid molecule is selectedfrom the group consisting of: (i) a nucleotide sequence as set forth inSEQ ID NO:1 or a nucleotide sequence having at least about 30%similarity thereto or a nucleotide sequence capable of hybridizing toSEQ ID NO:1 or its complementary form under low stringency conditions;(ii) a nucleotide sequence as set forth in SEQ IUD NO:2 or a nucleotidesequence having at least about 30% similarity thereto or a nucleotidesequence capable of hybridizing to SEQ ID NO:2 or its complementary formunder low stringency conditions; (iii) a nucleotide sequence as setforth in SEQ ID NO:3 or a nucleotide sequence having at least about 30%similarity thereto or a nucleotide sequence capable of hybridizing toSEQ ID NO:3 or its complementary form under low stringency conditions;(iv) a nucleotide sequence as set forth in SEQ ID NO:4 or a nucleotidesequence having at least about 30% similarity thereto or a nucleotidesequence capable of hybridizing to SEQ ID NO:4 or its complementary formunder low stringency conditions; (v) a nucleotide sequence as set forthin SEQ ID NO:5 or a nucleotide sequence having at least about 30%similarity thereto or a nucleotide sequence capable of hybridizing toSEQ ID NO:5 or its complementary form under low stringency conditions;and (vi) a nucleotide sequence as set forth in SEQ ID NO:6 or anucleotide sequence having at least about 30% similarity thereto or anucleotide sequence capable of hybridizing to SEQ ID NO:6 or itscomplementary form under low stringency conditions.
 2. The isolatednucleic acid molecule of claim 1 wherein the nucleic acid moleculecomprises the nucleotide sequence set forth in SEQ ID NO:1.
 3. Theisolated nucleic acid molecule of claim 1 wherein the nucleic acidmolecule comprises the nucleotide sequence set forth in SEQ ID NO:2. 4.The isolated nucleic acid molecule of claim 1 wherein the nucleic acidmolecule comprises the nucleotide sequence set forth in SEQ ID NO:3. 5.The isolated nucleic acid molecule of claim 1 wherein the nucleic acidmolecule comprises the nucleotide sequence set forth in SEQ ID NO:4. 6.The isolated nucleic acid molecule of claim 1 wherein the nucleic acidmolecule comprises the nucleotide sequence set forth in SEQ ID NO:5. 7.The isolated nucleic acid molecule of claim 1 wherein the nucleic acidmolecule comprises the nucleotide sequence set forth in SEQ ID NO:6. 8.An isolated molecule comprising a sequence of nucleotides or amino acidsencoded by a nucleic acid molecule which is differentially expressed inhypothalamus, liver and/or pancreatic tissue in obese animals comparedto lean animals or in fasted animals compared to fed animals or indiabetic animals compared to non-diabetic animals wherein the isolatedmolecule is encoded by a nucleic acid molecule selected from the groupconsisting of: (i) a nucleic acid molecule as set forth in SEQ ID NO: 1or a nucleotide sequence having at least about 30% similarity to SEQ IDNO:1 or a nucleotide sequence capable of hybridizing to SEQ ID NO:1 orits complementary form under low stringency conditions; (ii) a nucleicacid molecule as set forth in SEQ ID NO:2 or a nucleotide sequencehaving at least about 30% similarity to SEQ ID NO:2 or a nucleotidesequence capable of hybridizing to SEQ ID NO:2 or its complementary formunder low stringency conditions; (iii) a nucleic acid molecule as setforth in SEQ ID NO:3 or a nucleotide sequence having at least about 30%similarity to SEQ ID NO:3 or a nucleotide sequence capable ofhybridizing to SEQ ID NO:3 or its complementary form under lowstringency conditions; (iv) a nucleic acid molecule as set forth in SEQID NO:4 or a nucleotide sequence having at least about 30% similarity toSEQ ID NO:4 or a nucleotide sequence capable of hybridizing to SEQ IDNO:4 or its complementary form under low stringency conditions; (v) anucleic acid molecule as set forth in SEQ ID NO:5 or a nucleotidesequence having at least about 30% similarity to SEQ ID NO:5 or anucleotide sequence capable of hybridizing to SEQ ID NO:5 or itscomplementary form under low stringency conditions; and (vi) a nucleicacid molecule as set forth in SEQ ID NO:6 or a nucleotide sequencehaving at least about 30% similarity to SEQ ID NO:6 or a nucleotidesequence capable of hybridizing to SEQ ID NO:6 or its complementary formunder low stringency conditions.
 9. The isolated molecule of claim 8wherein the molecule is a protein.
 10. The isolated protein of claim 9encoded by a nucleotide sequence set forth in SEQ ID NO:1.
 11. Theisolated protein of claim 9 encoded by a nucleotide sequence set forthin SEQ ID NO:2.
 12. The isolated protein of claim 9 encoded by anucleotide sequence set forth in SEQ ID NO:3.
 13. The isolated proteinof claim 9 encoded by a nucleotide sequence set forth in SEQ ID NO:4.14. The isolated protein of claim 9 encoded by a nucleotide sequence setforth in SEQ ID NO:5.
 15. The isolated protein of claim 9 encoded by anucleotide sequence set forth in SEQ ID NO:6.
 16. An isolated proteinencoded by a nucleic acid molecule which molecule is differentiallyexpressed in hypothalamus, pancreas or liver tissue of obese animalscompared to lean animals or a derivative, homolog, analog, chemicalequivalent or mimetic thereof, wherein said protein is selected from thegroup consisting of: (i) a protein encoded by a nucleotide sequencesubstantially as set forth in SEQ ID NO:1 or a derivative, homolog oranalog thereof or a sequence encoding an amino acid sequence having atleast about 30% similarity to this sequence or a derivative, homolog,analog, chemical equivalent or mimetic of said protein, (ii) a proteinencoded by a nucleotide sequence substantially as set forth in SEQ IDNO:2 or a derivative, homolog or analog thereof or a sequence encodingan amino acid sequence having at least about 30% similarity to thissequence or a derivative, homolog, analog, chemical equivalent ormimetic of said protein, (iii) a protein encoded by a nucleotidesequence substantially as set forth in SEQ ID NO:3 or a derivative,homolog or analog thereof or a sequence encoding an amino acid sequencehaving at least about 30% similarity to this sequence or a derivative,homolog, analog, chemical equivalent or mimetic of said protein, (iv) aprotein encoded by a nucleotide sequence substantially as set forth inSEQ ID NO:4 or a derivative, homolog or analog thereof or a sequenceencoding an amino acid sequence having at least about 30% similarity tothis sequence or a derivative, homolog, analog, chemical equivalent ormimetic of said protein, (v) a protein encoded by a nucleotide sequencesubstantially as set forth in SEQ ID NO:5 or a derivative, homolog oranalog thereof or a sequence encoding an amino acid sequence having atleast about 30% similarity to this sequence or a derivative, homolog,analog, chemical equivalent or mimetic of said protein, (vi) a proteinencoded by a nucleotide sequence substantially as set forth in SEQ IDNO:6 or a derivative, homolog or analog thereof or a sequence encodingan amino acid sequence having at least about 30% similarity to thissequence or a derivative, homolog, analog, chemical equivalent ormimetic of said protein, (vii) a protein encoded by a nucleic acidmolecule capable of hybridizing to the nucleotide sequence as set forthin SEQ ID NO:1 or a derivative, homolog or analog thereof under lowstringency conditions, (viii) a protein encoded by a nucleic acidmolecule capable of hybridizing to the nucleotide sequence as set forthin SEQ ID NO:2 or a derivative, homolog or analog thereof under lowstringency conditions, (ix) a protein encoded by a nucleic acid moleculecapable of hybridizing to the nucleotide sequence as set forth in SEQ IDNO:3 or a derivative, homolog or analog thereof under low stringencyconditions, (x) a protein encoded by a nucleic acid molecule capable ofhybridizing to the nucleotide sequence as set forth in SEQ ID NO:4 or aderivative, homolog or analog thereof under low stringency conditions,(xi) a protein encoded by a nucleic acid molecule capable of hybridizingto the nucleotide sequence as set forth in SEQ ID NO:5 or a derivative,homolog or analog thereof under low stringency conditions, and (xii) aprotein encoded by a nucleic acid molecule capable of hybridizing to thenucleotide sequence as set forth in SEQ ID NO:6 or a derivative, homologor analog thereof under low stringency conditions.
 17. A method formodulating expression of one or more of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and/or AGT-204 in a mammal, said method comprisingcontacting AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204with an effective amount of a modulator of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and/or AGT-204 expression for a time and underconditions sufficient to up-regulate or down-regulate or otherwisemodulate expression of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601and/or AGT-204.
 18. A method of modulating activity of AGT-109, AGT-407,AGT-408, AGT-409, AGT-601 and/or AGT-204 in a mammal, said methodcomprising administering to said mammal a modulating effective amount ofa molecule for a time and under conditions sufficient to increase ordecrease AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204activity.
 19. A method of treating a mammal suffering from a conditioncharacterized by one or more symptoms of obesity, anorexia, diabetesand/or energy imbalance, said method comprising administering to saidmammal an effective amount of an agent for a time and under conditionssufficient to modulate the expression of AGT-109, AGT-407, AGT-408,AGT-409, AGT-601 and/or AGT-204 or sufficient to modulate the activityof AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204.
 20. Amethod of treating a mammal suffering from a disease conditioncharacterized by one or more symptoms of obesity, anorexia, diabetes orenergy imbalance, said method comprising administering to said mammal aneffective amount of AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/orAGT-204 or AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204.21-23. (canceled)
 24. A composition comprising a modulator of AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204 expression or AGT-109,AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204 activity and one ormore pharmaceutically acceptable carriers and/or diluents.
 25. A methodfor detecting AGT-109, AGT-407, AGT-408, AGT-409, AGT-601 and/or AGT-204or a derivative or homolog thereof in a biological sample from asubject, said method comprising contacting said biological sample withan antibody specific for AGT-109, AGT-407, AGT-408, AGT-409, AGT-601and/or AGT-204 or their antigenic derivatives or homologs for a time andunder conditions sufficient for a complex to form, and then detectingsaid complex.